Novel fab fragment libraries and methods for their use

ABSTRACT

The present invention provides Fab libraries and methods for using the Fab libraries to obtain antibodies against a target. The Fab library of the invention contains at least 10 9  different Fabs, and in some embodiments, at least 10 10  different Fabs. The Fab libraries of the invention are used to isolate polyclonal or monoclonal Fabs that bind with high specificity to targets.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 11/862,791, filed on Sep. 27, 2007, which is a continuationapplication of U.S. application Ser. No. 09/988,899, filed on Nov. 19,2001, which is a continuation of international application no.PCT/US00/13682, filed on May 18, 2000 which claims priority to Europeanapplication no. 99201558.6, filed on May 18, 1999. The contents of theseapplications are herein incorporated by reference in their entireties.

This invention relates in general to phage display libraries of humanFab fragments, and methods using the Fab fragment libraries to isolatehigh affinity antibodies. Especially, the invention relates topolynucleotides encoding a Fab library, Fab libraries, and methods fordesigning, constructing and selecting from Fab libraries.

Display on filamentous phage in combination with selection forms apowerful tool for the identification of peptide- or protein-based drugs(Winter et al., 1994; Clackson et al., 1994). Of these, antibodies areespecially of interest, due to their capacity to recognize a variety oftargets with high specificity and affinity. Particularly the use ofpartial or complete human antibodies, which elicit no or minimal immuneresponse when administered to patients, is yielding an increasing listof FDA-approved protein-based drugs (Holliger et al. 1998). Phagedisplay technology enables the generation of large repertoires of humanantibodies (Marks et al., 1991, Hoogenboom et al., 1992; Griffiths etal., 1993; Vaughan et al., 1996), and biopanning procedures permit theselection of individual antibodies with a desired specificity.

Key to the success of the technology were two critical observations: (i)the expression of functional antibody fragments by secretion into theperiplasm of E. coli (Better et al., 1988; Skerra et al., 1988), and,(ii) the rapid access to variable region gene pools by the polymerasechain reaction (Larrick et al., 1989; Ward et al., 1989; Marks et al.,1991). For the construction of antibody libraries, V-genes are amplifiedfrom B-cell cDNA and heavy and light chain genes are randomly combinedand cloned to encode a combinatorial library of single-chain Fv (scFv)or Fab antibody fragments (Marks et al., 1991; Clackson et al. 1991;Persson et al., 1991; Orum et al., 1993). The natural primary(unselected) antibody repertoire within B-cells contains a large arrayof antibodies that recognize a variety of antigens; this array can becloned as a ‘naVve’ repertoire of rearranged genes, by harvesting theV-genes from the IgM mRNA of B-cells of unimmunized human donors,isolated from peripheral blood lymphocytes (Marks et al., 1991), bonemarrow or tonsils (Vaughan et al., 1996), or from similar animal sources(Gram et al., 1992). This procedure provides access to antibodies thathave not yet encountered antigen, although the frequency of thosegenuine ‘germline’ antibodies will depend heavily on the source ofB-cells (Klein et al., 1997). A single ‘naïve’ library, if sufficientlylarge and diverse, can indeed be used to generate antibodies to a largepanel of antigens, including self, non-immunogenic and relatively toxicantigens (Griffiths et al., 1993; Marks et al., 1991). In a differentapproach, antibodies may be built artificially, by in vitro assembly ofV-gene segments and D/J segments, yielding ‘synthetic’ antibodies(Hoogenboom et al., 1992). A major drawback of these procedures is thatfrom the initial ‘naive’ and ‘synthetic’ libraries, only moderateaffinity antibodies were isolated (Marks et al., 1991; Nissim et al.,1994). Over the last few years more efficient techniques have beendeveloped to build larger libraries of antibody fragments, usingsophisticated in vivo recombination methods (Griffiths et al., 1993) orbrute force cloning procedures (Vaughan et al., 1996; Sheets et al.,1998). Such large libraries have yielded a greater number of humanantibodies per antigen tested, with an average much higher affinity (upto sub-nanomolar). However, technical restrictions on the size oflibraries that may be obtained or handled in selection, the loss oflibrary diversity upon library amplification, and the relatively longdown-stream analysis path of the selected antibodies, i.e., large scaleaffinity analysis, have limited the spread of these libraries as generictools in antibody generation.

Most large libraries made to date use the single chain format fordisplay on phage (Vaughan et al., 1996; Sheets et al. 1998). One reportdescribed the use of a human naive Fab library on phage (not permittingimmediate screening of selected soluble Fab fragments) (Griffiths etal., 1994). scFv's have the tendency to form dimers and higher ordermultimers in a clone-dependent and relatively unpredictable way(Weidner, et al. 1992; Holliger, et al. 1993; Marks et al., 1993). As aconsequence, the affinity assay used (such as BIAcore analysis) oftennecessitates purification of the selected antibody fragments. Forexample, ranking for off-rates using BIAcore is not easily possible withunpurified scFv fragments; the monomeric fraction of selected scFvclones first needs to be purified by affinity chromatography andgel-filtration (Sheets et al., 1998; Schier et al., 1996).

As was postulated and observed by Griffiths and colleagues (Griffiths etal., 1994), the size of the antibody library dictates the probability ofthe selection of high affinity antibodies to the antigen. Comparison ofthe first naVve scFv repertoire containing 2.9×10⁷ clones (Marks et al.,1991), with a recently constructed scFv repertoire of approximately 10¹⁰clones (Vaughan et al., 1996; Sheets et al. 1998), confirms thispostulation: increasing the library size 500-fold resulted inapproximately 100-fold higher affinities. This increase is caused bylowering the off-rates from 10⁻¹-10⁻² s⁻¹ for fragments selected fromthe smaller sized library to 10⁻³-10⁻⁴ s⁻¹ for those from the largerlibrary.

It is an object of the invention to create a Fab library that is avaluable source of antibodies for many different targets, and which willplay a vital role in target discovery and validation in the area offunctional genomics.

The invention provides a plurality of polynucleotides encoding a Fablibrary comprising a plurality of vector wherein the vector comprises:

-   -   a first and second cloning region, wherein        -   each cloning region comprises at least one, for the vector            unique, restriction enzyme cleavage site,        -   each cloning region being 5′ flanked by a ribosome binding            site and a signal sequence,    -   a polynucleotide encoding an anchor region, located 3′ of the        second cloning region,    -   a first and a second plurality of variable polynucleotides,        -   each encoding a complete antibody variable region or part of            an antibody variable region, possibly followed by a complete            antibody constant region or part of an antibody constant            region,        -   the first plurality of variable polynucleotides being cloned            into the vector at the restriction enzyme cleavage site(s)            of the first cloning region,        -   the second plurality of variable polynucleotides being            cloned into the vector at the restriction enzyme cleavage            site(s) of the second cloning region.

It is to be understood that the term “for the vector unique restrictionenzyme cleavage site” refers to the presence of one of such arestriction site in the vector sequence, without taking into account thepossible presence of such a site on the above-mentioned first and/orsecond polynucleotides encoding a complete antibody variable region orpart of an antibody variable region, possibly followed by a completeantibody constant region or part of an antibody constant region. Thesaid first and second polynucleotides may comprise restriction sitesidentical to the “unique” site. This means that the said restrictionsite was “unique” before both first and second polynucleotide sequenceswere cloned into the vector.

The first and second variable polynucleotides are preferably cloned inthe cloning region in a predetermined orientation. Therefore, in casethe cloning region comprises a single unique restriction site, this siteis preferably of such a type that non-identical restriction ends aregenerated, such as e.g, generated by the restriction enzyme SfiI.However, the cloning region may comprise two or more unique restrictionsites, so that the variable polynucleotides can be conveniently clonedas a restriction fragment that has the corresponding ends.

Preferably, in the vector according to the invention, the first andsecond cloning regions, both ribosomal binding sites, signal sequencesand the anchor sequence are part of a single polylinker sequence. Bothcloning regions may therefore be part of a single cassette, comprisingthe first cloning region, 5′ flanked by a ribosomal binding site and asignal sequence, lying adjacent to the second cloning region, also 5′flanked by its corresponding ribosomal binding site and a signalsequence, and 3′ flanked by the anchor sequence.

Preferably, the first plurality of variable polynucleotides are V_(L)polynucleotides, and the second plurality of variable polynucleotidesare V_(H) polynucleotides. More preferably, the V_(L) polynucleotidesare V₆ polynucleotides, V₆C₆ polynucleotides, V₈ polynucleotides, V₈C₈polynucleotides, a mixture of V₆ and V₈ polynucleotides, or a mixture ofV₆C₆ and V₈C₈ polynucleotides.

In another embodiment of the polynucleotides according to the invention,the vector further comprises a tag for purification or detection of anantibody, said tag for purification of the antibody preferablycomprising a poly-histidine tail; the tag for detection of the antibodyis preferably a c-myc-derived tag.

In another embodiment of the polynucleotides according to the invention,the vector further comprises an amber stop codon located between thesecond variable polynucleotide and the anchor protein.

In still another embodiment of the polynucleotides according to theinvention, the vector further comprises a C_(H1) domain located betweenthe second variable polynucleotide and the anchor protein, the C_(H1)domain preferably being a human gamma-1 C_(H1) domain.

“Anchor protein” is defined as a protein or part thereof that can atleast partially be accommodated in the outer coat of a particlegenerated by an organism expressing the library, such as a phage orvirus particle, or in the outer coat of an organism itself, in case theorganism itself expresses the library. The outer coat is herein definedas the structure of a cell, virus or phage particle defining the outersurface thereof. In case of a phage or phagemid expressing the library,the anchor protein may be a coat protein, such as the gene III product.However, other systems, known to the skilled person, may be used toobtain a library according to the present invention. Therefore, e.g.,transmembrane proteins, or the transmembrane domain thereof, may becontemplated to be used as anchor protein in eukaryotic expressionsystems. In the invention, the anchor protein may be fused to anantibody variable region or part thereof, resulting in the presentationof the said variable region to the outer environment of the organism,the region being anchored in its outer coat.

In a preferred embodiment of the polynucleotides according to theinvention, the anchor protein is a minor coat protein III of afilamentous phage f_(d).

In one embodiment of the invention, the polynucleotides according to theinvention, and therefore the Fab library, encodes at least 10⁹ differentFabs. In another embodiment of the invention, the Fab library of theinvention encodes at least 10¹⁰ different Fabs. In still anotherembodiment of the invention, the Fab library encodes at least 3.7×10¹⁰different Fabs. In still another embodiment of the invention, the Fablibrary encodes 10⁹ to 3.7×10¹° different Fabs.

Further, the invention provides a Fab library, comprising

-   -   a plurality of vectors as defined above,    -   the second cloning region in each vector forming a fusion        polynucleotide encoding a plurality of fusion proteins,    -   a plurality of capsid particles, wherein the plurality of vector        containing the first and second pluralities of variable        polynucleotides is packaged into the capsid particles, wherein    -   at least some of the capsid particles display the fusion protein        encoded by the vector packaged into the capsid on the surface of        the capsid.        Further the invention relates to a method of making a plurality        of polynucleotides encoding a Fab library, comprising the steps        of:    -   amplifying a first plurality of variable polynucleotides with a        first set of primers,    -   amplifying a second plurality of variable polynucleotides with a        second set of primers,        -   wherein each set of primers comprises oligonucleotides            designed to be homologous to the 5′ and 3′ end of variable            polynucleotides encoding antibody variable regions or parts            thereof, such that they can be used to amplify variable            polynucleotide pools from natural or synthetic sources of            genes while retaining all or part of the antibody's antigen            combining site;    -   cloning the first and second plurality of variable        polynucleotides into a plurality of vectors,        -   wherein the vector comprises:            -   a first and a second cloning region, wherein                -   each cloning region comprises at least one, for the                    vector unique, restriction enzyme cleavage site,                -   each cloning region being 5′ flanked by a ribosome                    binding site and a signal sequence,            -   a polynucleotide encoding an anchor region, located 3′                of the second cloning region,        -   wherein the first plurality of variable polynucleotides is            cloned into the restriction enzyme cleavage site(s) of the            first cloning region of the vector and the second plurality            of variable polynucleotides into the restriction enzyme            cleavage site(s) of the second cloning region of the vector.

In one embodiment, the method of constructing the Fab library comprisesthe steps of: amplifying a plurality of variable gene pools with a setof the primers, wherein the primers comprise oligonucleotides designedto be homologous to the 5′ and 3′ end of variable polynucleotidesencoding antibody variable regions or parts thereof, such that they canbe used to amplify variable polynucleotide pools from natural orsynthetic sources of genes while retaining all or part of the antibody'santigen combining site; cloning the amplified variable gene pools into avector with a two-step procedure to obtain a Fab library; wherein thevector comprises a phage or phagemid vector which will accommodateexpression of the cloned antibody variable polynucleotides as antibodyFab fragments, wherein one of the two antibody chains is fused to one ofthe phage coat proteins (e.g., geneIII product).

In one embodiment, the BACK primers were designed to have at the mostthree mutations in a total of twentyone to twentythree nucleotides whencompared to the human germline gene segment region they would have tobind to, but with at least 3 homologous residues towards the 3′ site ofthe oligonucleotide. This set of olignucleotides will recogniseapproximately 90% of human germline gene segments and as such provideaccess to most of the present diversity of the B-cells in non-immunizedsources. In another embodiment, the heavy chain primers should end with‘GG’ to ensure stable binding at high annealing temperatures (at least55EC). Similarly the VkappaBACK primers and most of the VlambdaBACKprimers will be designed to preferentially end in ‘CC’. In an alternateembodiment, the primers consist of the sequences in FIG. 2.

The invention also provides methods for obtaining antibodies specific toan antigen from the Fab library. In certain embodiments, the methods ofthe invention allow a rapid initial screen of off-rates using the Fablibraries of the invention. In alternate embodiments, the methods of thepatent are used to screen off-rates for a large series of antigenspecific Fabs using the Fab libraries of the invention.

The present invention also relates to isolated antibodies specific to anantigen of choice, and their corresponding nucleic acids, that areisolated from the Fab libraries of the invention. In an alternativeembodiment, these isolated antibodies are high affinity antibodies. Theantibodies may be used as research reagents or as therapeutic products.The antibodies of the invention will be ideal for investigating thenature and localization of their targets, and the antibodies can be usedto purify the target. Thus, the antibodies of the invention will beimportant for target validation and target discovery in the area offunctional genomics.

The invention also relates to a vector as is defined above, comprisingone of the first and one of the second plurality of the variablepolynucleotides cloned into the first and second cloning regionrespectively.

The present invention further relates to host cells containing the Fablibraries of the invention or the polynucleotides that encode the Fablibraries of the invention.

In one aspect the invention involves linking the desired specificbinding pair member, such as an antibody molecule, to a phage coatprotein. By then enriching for the specific binding pair member, such asby affinity techniques, for example, the DNA which encodes the specificbinding pair member is also enriched and may then be isolated. The DNAso obtained may then be cloned and expressed in other systems, yieldingpotentially large quantities of the desired specific binding pairmember, or may be subjected to sequencing and further cloning andgenetic manipulations prior to expression.

Typically the target for the specific binding pair member, e.g., anantigen or hapten when the specific binding pair member is an antibody,is known, and the methods herein provide a means for creating and/oridentifying a specific binding pair member which specifically binds thetarget of interest. Thus, when the protein is an antibody the presentinvention provides a novel means for producing antibodies, particularlymonoclonal antibodies, with specificity for predetermined targets,thereby circumventing the laborious, time-consuming and oftenunpredictable process of conventional monoclonal antibody technology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Phagemid vector pCES1 for display of antibody Fab fragments.Schematic representation (A) and polylinker region (SEQ ID NOS: 5-8) (B)of pCES1. The polylinker region comprises two signal sequences (‘S’;pelB and the geneIII leader sequence), the C6 domain, ribosome bindingsite (rbs), CH1 domain, hexa histidine tag (H6) and a c-myc derivedsequence. Variable domain genes can be cloned as ApaLI-XhoI or ApaLI-Ascfragments (for VL or VLCL respectively) and SfiI/PstI-BstEII orSfiI-NotI fragments (for VH or VHCH1 respectively. The amber stop codon(*) between the antibody genes and bacteriophage gene III enables theproduction of soluble Fab fragments in a non-suppressor strain of E.coli. Expression of the bicistronic operon is under control of the LacZpromotor (pLacZ).

FIG. 1B. shows polylinker region of the phagemid vector pCES1 (SEQ IDNO:6)

FIG. 2A. lists oligonucleotide primers (SEQ ID Nos:9-21) used forconstruction of the library.

FIG. 2B. lists oligonucleotide primers (SEQ ID Nos:22-27) forconstruction of the library.

FIG. 2C. lists oligonucleotide primers (SEQ ID Nos:28-38) forconstruction of the library.

FIG. 2D. lists oligonucleotide primers (SEQ ID Nos:39-50) forconstruction of the library.

FIG. 2E. lists oligonucleotide primers (SEQ ID Nos:51-60) forconstruction of the library.

FIG. 2F. lists oligonucleotide primers (SEQ ID Nos:61-71) forconstruction of the library.

The term “active” refers to those forms of the polypeptide which retainthe biologic and/or immunologic activities of any naturally occurringpolypeptide.

The term “activated” cells as used in this application are those whichare engaged in extracellular or intracellular membrane trafficking,including the export of neurosecretory or enzymatic molecules as part ofa normal or disease process.

The term ‘antibody’ means an immunoglobulin whether natural or partly orwholly synthetically produced. The term also covers any protein orpolypeptide having a binding domain which is homologous to animmunoglobulin binding domain. These proteins can be derived fromnatural sources, or partly or wholly synthetically produced. Exampleantibodies are the immunoglobulin isotypes and the Fab, scFv, Fv, dAab,VHH, Fd fragments.

The term ‘antibody polypeptide dimer’ means an association of twopolypeptide chain components of an antibody, capable of binding anantigen. Thus, it may be one arm of an antibody consisting of a heavychain and a light chain, it may be a Fab fragment consisting of V_(L),V_(H), C_(L) and C_(H1) antibody domains, or an Fv fragment consistingof a V_(L) domain and a V_(H) domain.

The term ‘capsid’ means a replicable genetic display package, with orwithout the genetic information. The capsids display a member of aspecific binding pair at its surface. The package may be a population ofbacteriophages which display an antigen binding domain, e.g., a Fab, atthe surface of some or all of the capsids within the population. Thistype of package has been called a phage antibody (pAb).

The term ‘C_(H1) domain’ means the first constant region of the heavychain of an antibody or part thereoff or extended with aminoacids fromthe hinge regions as to allow pairing of the expressed (VH)CH1 fragmentwith the antibody's light chain, and possible disulphide-bridgeformation. This may be the CH1 domain of a human antibody of isotypegamma-1.

A “component part of an antibody antigen-binding site” may be orcorrespond to a polypeptide chain component, e.g., a V_(H) or a V_(L)domain. However, it may be a CDR, or a V_(L) sequence plus CDR of aV_(H), a V_(H) sequence plus CDR of a V_(L), a V_(H) plus V_(L) sequencelacking only a CDR, and so on. The proviso is that the first and secondcomponent parts of an antigen-binding site of an antibody must incombination (together) form an antigen-binding site. Thus, if the secondcomponent part of an antigen-binding site of a non-human antibodyspecific for an antigen of interest is a CDR, then the first componentpart of an antigen-binding site of a human antibody will comprise theremainder of a V_(H) and V_(L) region required to form a antigen-bindingsite (with or without associated antibody constant domains (in a Fabformat), or with or without a linker peptide sequence (in a Fv format).The second component part of an antigen-binding site of a non-humanantibody may comprise a V_(L) domain plus part of a V_(H) domain, thatpart being one or more CDRs, for instance, perhaps CDR3. In such case,the first component part of an antigen-binding site of a human antibodywould comprise the remainder of a V_(H) sequence which in combinationwith the second component part forms an antigen-binding site. Of course,the converse situation holds and the person skilled in the art will beable to envisage other combinations of first and second component partswhich together form an antigen-binding site.

The term ‘conditionally defective’ means a gene which does not express aparticular polypeptide under one set of conditions, but expresses itunder another set of conditions. An example, is a gene containing anamber mutation expressed in non-suppressing or suppressing hostsrespectively. Alternatively, a gene may express a protein or polypeptidewhich is defective under one set of conditions, but not under anotherset. An example is a gene with a temperature sensitive mutation.

The term “derivative” refers to polypeptides chemically modified by suchtechniques as ubiquitination, labeling (e.g., with radionuclides orvarious enzymes), pegylation (derivatization with polyethylene glycol)and insertion or substitution by chemical synthesis of amino acids suchas ornithine, which do not normally occur in human proteins.

The term ‘domain’ means a part of a protein or polypeptide that isfolded within itself and independently of other parts of the sameprotein or polypeptide and independently of a complementary bindingmember.

The term ‘eluant’ means a solution used to breakdown the linkage betweentwo molecules. The linkage can be a non-covalent or covalent bond(s).The two molecules can be members of a sbp.

The term ‘expression modulating fragment,’ EMF, means a series ofnucleotides which modulates the expression of an operably linked ORF oranother EMF.

As used herein, a sequence is said to ‘modulate the expression of anoperably linked sequence’ when the expression of the sequence is alteredby the presence of the EMF. EMFs include, but are not limited to,promoters, and promoter modulating sequences (inducible elements). Oneclass of EMFs are fragments which induce the expression or an operablylinked ORF in response to a specific regulatory factor or physiologicalevent.

The term “Fab” refers to antibody fragments including fragments whichcomprise two N-terminal portions of the heavy chain polypeptide joinedby at least one disulfide bridge in the hinge region and two completelight chain polypeptides, where each light chain is complexed with oneN-terminal portion of a heavy chain. Fab also includes Fab fragmentswhich comprise all or a large portion of a light chain polypeptide(e.g., V_(L)C_(L)) complexed with the N-terminal portion of a heavychain polypeptide (e.g., V_(H)C_(H1)).

The term “Fab library” refers to a collection of Fab polynucleotidesequences within clones; or a genetically diverse collection of Fabpolypeptides displayed on rgdps capable of selection or screening toprovide an individual Fab polypeptide or a mixed population of Fabpolypeptides.

The term ‘folded unit’ means a specific combination of an alpha-helixand/or beta-strand and/or beta-turn structure. Domains and folded unitscontain structures that bring together amino acids that are not adjacentin the primary structure.

The term ‘genetically diverse population’ means antibodies orpolypeptide components thereof, this is referring not only to diversitythat can exist in the natural population of cells or organisms, but alsodiversity that can be created by artificial mutation in vitro or invivo. Mutation in vitro may for example, involve random mutagenesisusing oligonucleotides having random mutations of the sequence desiredto be varied. In vivo mutagenesis may for example, use mutator strainsof host microorganisms to harbour the DNA (see Example 38 of WO92/01047). The words “unique population” may be used to denote aplurality of e.g., polypeptide chains, which are not genetically diversei.e., they are all the same. A restricted population is one which isdiverse but less so than the full repertoire of an animal. The diversitymay have been reduced by prior selection, e.g., using antigen bindingspecificity.

The term ‘helper phage’ means a phage which is used to infect cellscontaining a defective phage genome and which functions to complementthe defect. The defective phage genome can be a phagemid or a phage withsome function encoding gene sequences removed. Examples of helper phagesare M13KO7, M13KO7 gene III no. 3; and phage displaying or encoding abinding molecule fused to a capsid protein.

The term ‘homologs’ means polypeptides having the same or conservedresidues at a corresponding position in their primary, secondary ortertiary structure. The term also extends to two or more nucleotidesequences encoding the homologous polypeptides. Example homologouspeptides are the immunoglobulin isotypes.

The term “host cell” refers to a prokaryotic or eukaryotic cell intowhich the vectors of the invention may be introduced, expressed and/orpropagated. Typical prokaryotic host cells include various strains of E.coli. Typical eukaryotic host cells are yeast or filamentous fungi, ormammalian cells, such as Chinese hamster ovary cells, murine NIH 3t3fibroblasts, or human embryonic kidney 193 cells.

The term ‘immunoglobulin superfamily’ means a family of polypeptides,the members of which have at least one domain with a structure relatedto that of the variable or constant domain of immunoglobulin molecules.The domain contains two B-sheets and usually a conserved disulphide bond(see A. F. Williams and A. N. Barclay 1988 Ann. Rev Immunol. 6 381-405).Example members of an immunoglobulin superfamily are CD4, plateletderived growth factor receptor (PDGFR), intercellular adhesion molecule.(ICAM). Except where the context otherwise dictates, reference toimmunoglobulins and immunoglobulin homologs in this application includesmembers of the immunoglobulin superfamily and homologs thereof.

The term “infection” refers to the introduction of nucleic acids into asuitable host cell by use of a virus or viral vector.

The term “intermediate fragment” means a nucleic acid between 5 and 1000bases in length, and preferably between 10 and 40 bp in length.

The term “isolated” as used herein refers to a nucleic acid orpolypeptide separated not only from other nucleic acids or polypeptidesthat are present in the natural source of the nucleic acid orpolypeptide, but also from polypeptides, and preferably refers to anucleic acid or polypeptide found in the presence of (if anything) onlya solvent, buffer, ion, or other component normally present in asolution of the same. The terms “isolated” and “purified” do notencompass nucleic acids or polypeptides present in their natural source.

The term ‘mutator strain’ means a host cell which has a genetic defectwhich causes DNA replicated within it to be mutated with respect to itsparent DNA. Example mutator strains are NR9046mutD5 and NR9046 mut T1(See Example 38 of WO 92/01047).

The term “naturally occurring polypeptide” refers to polypeptidesproduced by cells that have not been genetically engineered andspecifically contemplates various polypeptides arising frompost-translational modifications of the polypeptide including, but notlimited to, acetylation, carboxylation, glycosylation, phosphorylation,lipidation and acylation.

The term ‘nucleotide sequence’ refers to a heteropolymer of nucleotidesor the sequence of these nucleotides. The terms ‘nucleic acid’ and‘polynucleotide’ are also used interchangeably herein to refer to aheteropolymer of nucleotides. Generally, nucleic acid segments providedby this invention may be assembled from fragments of the genome andshort oligonucleotide linkers, or from a series of oligonucleotides, orfrom individual nucleotides, to provide a synthetic nucleic acid whichis capable of being expressed in a recombinant transcriptional unitcomprising regulatory elements derived from a microbial or viral operon,or a eukaryotic gene.

The terms “oligonucleotide fragment” or a “polynucleotide fragment”,“portion,” or “segment” is a stretch of polypeptide nucleotide residueswhich is long enough to use in polymerase chain reaction (PCR) orvarious hybridization procedures to identify or amplify identical orrelated parts of mRNA or DNA molecules.

The terms “oligonucleotides” or “nucleic acid probes” are prepared basedon the polynucleotide sequences provided in the present invention.Oligonucleotides comprise portions of such a polynucleotide sequencehaving at least about 15 nucleotides and usually at least about 20nucleotides. Nucleic acid probes comprise portions of such apolynucleotide sequence having fewer nucleotides than about 6 kb,usually fewer than about 1 kb. After appropriate testing to eliminatefalse positives, these probes may, for example, be used to determinewhether specific mRNA molecules are present in a cell or tissue or toisolate similar nucleic acid sequences from chromosomal DNA as describedby Walsh et al. (Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).

The term “open reading frame,” ORF, means a series of nucleotidetriplets coding for amino acids without any termination codons and is asequence translatable into protein.

The term ‘phage vector’ means a vector derived by modification of aphage genome, containing an origin of replication for a bacteriophage,but not one for a plasmid.

The term ‘phagemid vector’ means a vector derived by modification of aplasmid genome, containing an origin of replication for a bacteriophageas well as the plasmid origin of replication.

The term “phenotype” refers to a physical (e.g., pigment, or cell shape)and/or metabolic property of a cell which can be measured or exploitedin some fashion and which is effected by the reporter gene.

The term ‘polylinker region’ means a polynucleotide that contains atleast two restriction enzyme sites that are unique in the vector thatcontains the polylinker region, i.e., these restriction sites are easilyused cloning sites in the vector.

A polypeptide “fragment,” “portion,” or “segment” is a stretch of aminoacid residues of at least about 5 amino acids, often at least about 7amino acids, typically at least about 9 to 13 amino acids, and, invarious embodiments, at least about 17 or more amino acids. To beactive, any polypeptide must have sufficient length to display biologicand/or immunologic activity.

The term “probes” includes naturally occurring or recombinant orchemically synthesized single- or double-stranded nucleic acids. Theymay be labeled by nick translation, Klenow fill-in reaction, PCR orother methods well known in the art. Probes of the present invention,their preparation and/or labeling are elaborated in Sambrook, J. et al.,1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols inMolecular Biology, John Wiley & Sons, New York N.Y., both of which areincorporated herein by reference in their entirety.

The term “purified” as used herein denotes that the indicated nucleicacid or polypeptide is present in the substantial absence of otherbiological macromolecules, e.g., polynucleotides, proteins, and thelike. In one embodiment, the polynucleotide or polypeptide is purifiedsuch that it constitutes at least 95% by weight, more preferably atleast 99.8% by weight, of the indicated biological macromoleculespresent (but water, buffers, and other small molecules, especiallymolecules having a molecular weight of less than 1000 daltons, can bepresent).

The term “recombinant,” when used herein to refer to a polypeptide orprotein, means that a polypeptide or protein is derived from recombinant(e.g., microbial or mammalian) expression systems. ‘Microbial’ refers torecombinant polypeptides or proteins made in bacterial or fungal (e.g.,yeast) expression systems. As a product, ‘recombinant microbial’ definesa polypeptide or protein essentially free of native endogenoussubstances and unaccompanied by associated native glycosylation.Polypeptides or proteins expressed in most bacterial cultures, e.g., E.coli, will be free of glycosylation modifications; polypeptides orproteins expressed in yeast will have a glycosylation pattern in generaldifferent from those expressed in mammalian cells.

The term ‘recombinant expression vehicle or vector’ refers to a plasmidor phage or virus or vector, for expressing a polypeptide from a DNA(RNA) sequence. An expression vehicle can comprise a transcriptionalunit comprising an assembly of (1) a genetic element or elements havinga regulatory role in gene expression, for example, promoters orenhancers, (2) a structural or coding sequence which is transcribed intomRNA and translated into protein, and (3) appropriate transcriptioninitiation and termination sequences. Structural units intended for usein yeast or eukaryotic expression systems preferably include a leadersequence enabling extracellular secretion of translated protein by ahost cell. Alternatively, where recombinant protein is expressed withouta leader or transport sequence, it may include an N-terminal methionineresidue. This residue may or may not be subsequently cleaved from theexpressed recombinant protein or polypeptide to provide a final product.

The term “recombinant expression system” means host cells which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit extrachromosomally.Recombinant expression systems as defined herein will expressheterologous polypeptides or proteins upon induction of the regulatoryelements linked to the DNA segment or synthetic gene to be expressed.This term also means host cells which have stably integrated arecombinant genetic element or elements having a regulatory role in geneexpression, for example, promoters or enhancers. Recombinant expressionsystems as defined herein will express polypeptides or proteinsendogenous to the cell upon induction of the regulatory elements linkedto the endogenous DNA segment or gene to be expressed. The cells can beprokaryotic or eukaryotic.

The term “recombinant variant” refers to any polypeptide differing fromnaturally occurring polypeptides by amino acid insertions, deletions,and substitutions, created using recombinant DNA techniques. Guidance indetermining which amino acid residues may be replaced, added or deletedwithout abolishing activities of interest, such as cellular trafficking,may be found by comparing the sequence of the particular polypeptidewith that of homologous peptides and minimizing the number of amino acidsequence changes made in regions of high homology.

Preferably, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements. Aminoacid substitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

“Insertions” or “deletions” are typically in the range of about 1 to 5amino acids. The variation allowed may be experimentally determined bysystematically making insertions, deletions, or substitutions of aminoacids in a polypeptide molecule using recombinant DNA techniques andassaying the resulting recombinant variants for activity.

Alternatively, where alteration of function is desired, insertions,deletions or non-conservative alterations can be engineered to producealtered polypeptides. Such alterations can, for example, alter one ormore of the biological functions or biochemical characteristics of thepolypeptides of the invention. For example, such alterations may changepolypeptide characteristics such as ligand-binding affinities,interchain affinities, or degradation/turnover rate. Further, suchalterations can be selected so as to generate polypeptides that arebetter suited for expression, scale up and the like in the host cellschosen for expression. For example, cysteine residues can be deleted orsubstituted with another amino acid residue in order to eliminatedisulfide bridges.

Alternatively, recombinant variants encoding these same or similarpolypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsin the polynucleotide sequence may be reflected in the polypeptide ordomains of other peptides added to the polypeptide to modify theproperties of any part of the polypeptide, to change characteristicssuch as ligand-binding affinities, interchain affinities, ordegradation/turnover rate.

The term ‘repertoire of artificially rearranged immunoglobulin genes’means a collection of nucleotide e.g., DNA, sequences derived wholly orpartly from a source other than the rearranged immunoglobulin sequencesfrom an animal. This may include for example, DNA sequences encoding VHdomains by combining unrearranged V segments with D and J segments andDNA sequences encoding VL domains by combining V and J segments. Part orall of the DNA sequences may be derived by oligonucleotide synthesis.

The term ‘repertoire of rearranged immunoglobulin genes’ means acollection of naturally occurring nucleotides e.g., DNA sequences whichencoded expressed immunoglobulin genes in an animal. The sequences aregenerated by the in vivo rearrangement of e.g., V, D and J segments forH chains and e.g., the V and J segments for L chains. Alternatively thesequences may be generated from a cell line immunised in vitro and inwhich the rearrangement in response to immunisation occursintracellularly. The word “repertoire” is used to indicate geneticdiversity.

The term ‘replicable genetic display package’ (Rgdp) means a biologicalparticle which has genetic information providing the particle with theability to replicate. The particle can display on its surface at leastpart of a polypeptide. The polypeptide can be encoded by geneticinformation native to the particle and/or artificially placed into theparticle or an ancestor of it. The displayed polypeptide may be anymember of a specific binding pair e.g., heavy or light chain domainsbased on an immunoglobulin molecule, an enzyme or a receptor etc. Theparticle may be a virus e.g., a bacteriophage such as fd or M13.

The term “reporter gene” refers to a nucleic acid which encodes aprotein or polypeptide that produces a phenotypic change in the hostcell that may be measured and/or used to separate host cells. Forexample, the reporter gene may encode a protein or polypeptide that hasflourescent properties, e.g., β-galactosidase, auto-fluorescent proteinGFP, etc.; or the reporter gene may encode a selectable marker, e.g.,antibiotic resistance; or an epitope that is expressed on the surface ofthe host cell.

The term ‘ribosome binding site’ means a polyribonucleotide that allowsa ribosome to select the proper initiation codon during the initiationof translation. In some prokaryotes, this polyribonucleotide is calledthe Shine-Dalgarno sequence, and the Shine-Delgarno sequence base pairswith the 16S RNA of the ribosome.

The term “secreted” protein or polypeptide refers to a protein orpolypeptide that is transported across or through a membrane, includingtransport as a result of signal sequences in its amino acid sequencewhen it is expressed in a suitable host cell. “Secreted” proteins orpolypeptides include without limitation proteins or polypeptidessecreted wholly (e.g., soluble proteins) or partially (e.g., receptors)from the cell in which they are expressed. “Secreted” proteins orpolypeptides also include without limitation proteins or polypeptideswhich are transported across the membrane of the endoplasmic reticulum.

The term ‘signal sequence’ means an amino acid sequence that is found atthe amino terminus of a polypeptide and directs transportation of thepolypeptide across or through a membrane. Signal sequences include aminoterminal polypeptides that are 13-36 residues long, and have a 7 to 13residue hydrophobic core flanked by several hydrophilic residues thatusually include one or more basic residues near the N-terminus.

The term “stringent” is used to refer to conditions that are commonlyunderstood in the art as stringent. An exemplary set of conditionsinclude a temperature of 60-70° C., (preferably about 65° C.) and a saltconcentration of 0.70 M to 0.80 M (preferably about 0.75M). Furtherexemplary conditions include, hybridizing conditions that (1) employ lowionic strength and high temperature for washing, for example, 0.015 MNaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.; (2) employ duringhybridization a denaturing agent such as formamide, for example, 50%(vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.

In instances wherein hybridization of deoxyoligonucleotides isconcerned, additional exemplary stringent hybridization conditionsinclude washing in 6×SSC/0.05% sodium pyrophosphate at 37EC (for 14-baseoligos), 48EC (for 17-base oligos), 55EC (for 20-base oligos), and 60EC(for 23-base oligos).

As used herein, “substantially equivalent” can refer both to nucleotideand amino acid sequences, for example a mutant sequence, that variesfrom a reference sequence by one or more substitutions, deletions, oradditions, the net effect of which does not result in an adversefunctional dissimilarity between the reference and subject sequences.Typically, such a substantially equivalent sequence varies from one ofthose listed herein by no more than about 20% (i.e., the number ofindividual residue substitutions, additions, and/or deletions in asubstantially equivalent sequence, as compared to the correspondingreference sequence, divided by the total number of residues in thesubstantially equivalent sequence is about 0.2 or less). Such a sequenceis said to have 80% sequence identity to the listed sequence. In oneembodiment, a substantially equivalent, e.g., mutant, sequence of theinvention varies from a listed sequence by no more than 10% (90%sequence identity); in a variation of this embodiment, by no more than5% (95% sequence identity); and in a further variation of thisembodiment, by no more than 2% (98% sequence identity). Substantiallyequivalent, e.g., mutant, amino acid sequences according to theinvention generally have at least 95% sequence identity with a listedamino acid sequence, whereas substantially equivalent nucleotidesequence of the invention can have lower percent sequence identities,taking into account, for example, the redundancy or degeneracy of thegenetic code. For the purposes of the present invention, sequenceshaving substantially equivalent biological activity and substantiallyequivalent expression characteristics are considered substantiallyequivalent. For the purposes of determining equivalence, truncation ofthe mature sequence (e.g., via a mutation which creates a spurious stopcodon) should be disregarded.

Nucleic acid sequences encoding such substantially equivalent sequences,e.g., sequences of the recited percent identities, can routinely beisolated and identified via standard hybridization procedures well knownto those of skill in the art.

The term ‘suppressible translational stop codon’ means a codon whichallows the translation of nucleotide sequences downstream of the codonunder one set of conditions, but under another set of conditionstranslation ends at the codon. Example of suppressible translationalstop codons are the amber, ochre and opal codons.

The term ‘tag’ means an extension of the antibody Fab fragment, forexample expressed at the carboxyterminus of the heavy chain, thatcomprises at least one amino acids but more typically five to fifteenamino acids, and that can be specifically recognised by an antibody orother binding ligand or binding matrix for the sequence. Tags may becombined in the same Fab. Examples are a stretch of five histidineresidues that can be recognised by specific antibodies and by definedimmobilised metal ions, and a stretch of the following 12 amino acids(EQKLISEEDLN) that are recognised by the 9E10 antibody (Marks et al.,1991).

The term ‘target’ means any molecule that is antigenic, e.g., can berecognized with reasonably specificity by an antibody from the Fablibrary.

The term ‘target element’ refers to a nucleic acid sequence that altersthe expression of the target gene. Target elements include, but are notlimited to, promoters, and promoter modulating sequences (inducibleelements). One class of target elements are fragments which induce theexpression in response to a specific regulatory factor or physiologicalevent.

The term “transfection” refers to the taking up of an expression vectorby a suitable host cell, whether or not any coding sequences are in factexpressed.

The term “transformation” means introducing DNA into a suitable hostcell so that the DNA is replicable, either as an extrachromosomalelement, or by chromosomal integration.

The term “universal set” refers to a set of nucleic acids, mostpreferably a set of oligonucleotides, which represent all possiblecombinations of sequence for a given length of nucleotides, e.g., all4096 insert oligonucleotides six nucleotides in length. In a preferredembodiment, the term universal set refers to the set of all possibleoligonucleotides of a given length, wherein one or more positions in theoligonucleotides are held constant (i.e., the same nucleotide is presentat this position in all members of the set).

As used herein, an ‘uptake modulating fragment,’ UMF, means a series ofnucleotides which mediate the uptake of a linked DNA fragment into acell. UMFs can be readily identified using known UMFs as a targetsequence or target motif with the computer-based systems describedbelow.

The presence and activity of a UMF can be confirmed by attaching thesuspected UMF to a marker sequence. The resulting nucleic acid moleculeis then incubated with an appropriate host under appropriate conditionsand the uptake of the marker sequence is determined. As described above,a UMF will increase the frequency of uptake of a linked marker sequence.

The term ‘vector’ refers to a plasmid or phage or virus or vector, forexpressing a polypeptide from a DNA (RNA) sequence. The vector cancomprise a transcriptional unit comprising an assembly of (1) a geneticelement or elements having a regulatory role in gene expression, forexample, promoters or enhancers, (2) a structural or coding sequencewhich is transcribed into mRNA and translated into protein, and (3)appropriate translation initiation and termination sequences. Structuralunits intended for use in yeast or eukaryotic expression systems mayinclude a leader sequence enabling extracellular secretion of translatedprotein by a host cell.

The term ‘V_(L) polynucleotides’ means polynucleotides encoding the CDRcontaining domains of some or all of the light chain genes from the V₆-and/or V₈-families.

The term ‘V_(H) polynucleotides’ means polynucleotides encoding the CDRcontaining domains of some or all of the heavy chain genes from theheavy chain gene family.

Each of the above terms is meant to encompasses all that is describedfor each, unless the context dictates otherwise.

The recombinant constructs of the present invention comprise a vector,such as a plasmid or viral vector, into which a nucleic acid(s) ofinterest may be inserted. The vector may further comprise regulatorysequences, including for example, a promoter, operably linked to thenucleic acid(s) of interest. Large numbers of suitable vectors andpromoters are known to those of skill in the art and are commerciallyavailable for generating the recombinant constructs of the presentinvention. The following vectors are provided by way of example.Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a,pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).

Methods which are well known to those skilled in the art can be used toconstruct vectors containing a polynucleotide of the invention andappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. See, for example, thetechniques described in Maniatis et al., Molecular Cloning A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y. (1989) and Ausubel et al.,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, N.Y. (1989).

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P, and trc.Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The polynucleotide of the invention isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the polynucleotide of the inventioncan encode a fusion protein including an N-terminal identificationpeptide imparting desired characteristics, e.g., stabilization orsimplified purification of expressed recombinant product.

Useful expression vectors for bacteria are constructed by inserting apolynucleotide of the invention together with suitable translationinitiation and termination signals, optionally in operable reading phasewith a functional promoter. The vector will comprise one or morephenotypic selectable markers and an origin of replication to ensuremaintenance of the vector and to, if desirable, provide amplificationwithin the host. Suitable prokaryotic hosts for transformation includeE. coli, Bacillus subtilis, Salmonella typhimurium and various specieswithin the genera Pseudomonas, Streptomyces, and Staphylococcus,although others may also be employed as a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacteria can comprise a selectable marker and bacterial origin ofreplication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, Wis.,USA). These pBR322 ‘backbone’ sections are combined with an appropriatepromoter and the structural sequence to be expressed.

The present invention further provides host cells containing the vectorsof the present invention, wherein the nucleic acid has been introducedinto the host cell using known transformation, transfection or infectionmethods. The host cell can be a higher eukaryotic host cell, such as amammalian cell, a lower eukaryotic host cell, such as a yeast cell, orthe host cell can be a prokaryotic cell, such as a bacterial cell.Introduction of the recombinant construct into the host cell can beeffected, for example, by calcium phosphate transfection, DEAF, dextranmediated transfection, or electroporation (Davis, L. et al., BasicMethods in Molecular Biology (1986)).

Any host/vector system can be used to identify one or more of the targetelements of the present invention. These include, but are not limitedto, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9cells, as well as prokaryotic host such as E. coli and B. subtilis. Themost preferred cells are those which do not normally express theparticular reporter polypeptide or protein or which expresses thereporter polypeptide or protein at low natural level.

The host of the present invention may also be a yeast or other fungi. Inyeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review see, Current Protocols in MolecularBiology, Vol. 2, Ed. Ausubel et al., Greene Publish. Assoc. & WileyInterscience, Ch. 13 (1988); Grant et al., Expression and SecretionVectors for Yeast, in Methods in Enzymology, Ed. Wu & Grossman, Acad.Press, N.Y. 153:516-544 (1987); Glover, DNA Cloning, Vol. II, IRL Press,Wash., D.C., Ch. 3 (1986); Bitter, Heterologous Gene Expression inYeast, in Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y.152:673-684 (1987); and The Molecular Biology of the YeastSaccharomyces, Eds. Strathern et al., Cold Spring Harbor Press, Vols. Iand II (1982).

The host of the invention may also be a prokaryotic cell such as E.coli, other enterobacteriaceae such as Serratia marescans, bacilli,various pseudomonads, or other prokaryotes which can be transformed,transfected, infected, etc. (i.e., a method exists for introducingnucleic acids to the host cell).

The present invention further provides host cells genetically engineeredto contain the polynucleotides of the invention. For example, such hostcells may contain nucleic acids of the invention introduced into thehost cell using known transformation, transfection or infection methods.The present invention still further provides host cells geneticallyengineered to express the polynucleotides of the invention, wherein suchpolynucleotides are in operative association with a regulatory sequenceheterologous to the host cell which drives expression of thepolynucleotides in the cell.

The host cell can be a higher eukaryotic host cell, such as a mammaliancell, a lower eukaryotic host cell, such as a yeast cell, or the hostcell can be a prokaryotic cell, such as a bacterial cell. Introductionof the recombinant construct into the host cell can be effected bycalcium phosphate transfection, DEAF, dextran mediated transfection, orelectroporation (Davis, L. et al., Basic Methods in Molecular Biology(1986)). The host cells containing one of polynucleotides of theinvention, can be used in conventional manners to produce the geneproduct encoded by the isolated fragment (in the case of an ORF) or canbe used to produce a heterologous protein under the control of the EMF.

Any host/vector system can be used to express one or more of the ORFs ofthe present invention. These include, but are not limited to, eukaryotichosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9 cells, as wellas prokaryotic host such as E. coli and B. subtilis. The most preferredcells are those which do not normally express the particular polypeptideor protein or which expresses the polypeptide or protein at low naturallevel. Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al., inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989), the disclosure of which is hereby incorporated byreference.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell tines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and also any necessary ribosome binding sites,polyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking nontranscribed sequences. DNAsequences derived from the SV40 viral genome, for example, SV40 origin,early promoter, enhancer, splice, and polyadenylation sites may be usedto provide the required nontranscribed genetic elements. Recombinantpolypeptides and proteins produced in bacterial culture are usuallyisolated by initial extraction from cell pellets, followed by one ormore salting-out, aqueous ion exchange or size exclusion chromatographysteps. Protein refolding steps can be used, as necessary, in completingconfiguration of the mature protein. Finally, high performance liquidchromatography (HPLC) can be employed for final purification steps.Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents.

A number of types of cells may act as suitable host cells for expressionof the protein. Mammalian host cells include, for example, monkey COScells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, humanepidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, othertransformed primate cell lines, normal diploid cells, cell strainsderived from in vitro culture of primary tissue, primary explants, HeLacells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.

Alternatively, it may be possible to produce the protein in lowereukaryotes such as yeast or in prokaryotes such as bacteria. Potentiallysuitable yeast strains include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous proteins. Potentially suitablebacterial strains include Escherichia coli, Bacillus subtilis,Salmonella typhimurium, or any bacterial strain capable of expressingheterologous proteins. If the protein is made in yeast or bacteria, itmay be necessary to modify the protein produced therein, for example byphosphorylation or glycosylation of the appropriate sites, in order toobtain the functional protein. Such covalent attachments may beaccomplished using known chemical or enzymatic methods.

In another embodiment of the present invention, cells and tissues may beengineered to express an endogenous gene comprising the polynucleotidesof the invention under the control of inducible regulatory elements, inwhich case the regulatory sequences of the endogenous gene may bereplaced by homologous recombination. As described herein, genetargeting can be used to replace a gene's existing regulatory regionwith a regulatory sequence isolated from a different gene or a novelregulatory sequence synthesized by genetic engineering methods. Suchregulatory sequences may be comprised of promoters, enhancers,scaffold-attachment regions, negative regulatory elements,transcriptional initiation sites, regulatory protein binding sites orcombinations of said sequences. Alternatively, sequences which affectthe structure or stability of the RNA or protein produced may bereplaced, removed, added, or otherwise modified by targeting, includingpolyadenylation signals. mRNA stability elements, splice sites, leadersequences for enhancing or modifying transport or secretion propertiesof the protein, or other sequences which alter or improve the functionor stability of protein or RNA molecules.

The targeting event may be a simple insertion of the regulatorysequence, placing the gene under the control of the new regulatorysequence, e.g., inserting a new promoter or enhancer or both upstream ofa gene. Alternatively, the targeting event may be a simple deletion of aregulatory element, such as the deletion of a tissue-specific negativeregulatory element. Alternatively, the targeting event may replace anexisting element; for example, a tissue-specific enhancer can bereplaced by an enhancer that has broader or different cell-typespecificity than the naturally occurring elements. Here, the naturallyoccurring sequences are deleted and new sequences are added. In allcases, the identification of the targeting event may be facilitated bythe use of one or more selectable marker genes that are contiguous withthe targeting DNA, allowing for the selection of cells in which theexogenous DNA has integrated into the host cell genome. Theidentification of the targeting event may also be facilitated by the useof one or more marker genes exhibiting the property of negativeselection, such that the negatively selectable marker is linked to theexogenous DNA, but configured such that the negatively selectable markerflanks the targeting sequence, and such that a correct homologousrecombination event with sequences in the host cell genome does notresult in the stable integration of the negatively selectable marker.Markers useful for this purpose include the Herpes Simplex Virusthymidine kinase (TK) gene or the bacterial xanthine-guaninephosphoribosyl-transferase (gpt) gene.

The gene targeting or gene activation techniques which can be used inaccordance with this aspect of the invention are more particularlydescribed in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461to Sherwin et al.; International Application No. PCT/US92/09627(WO93/09222) by Selden et al.; and International Application No.PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which isincorporated by reference herein in its entirety.

In general, techniques for preparing polyclonal and monoclonalantibodies as well as hybridomas capable of producing the desiredantibody are well known in the art (Campbell, A. M., MonoclonalAntibodies Technology Laboratory Techniques in Biochemistry andMolecular Biology, Elsevier Science Publishers, Amsterdam, TheNetherlands (1984); St. Groth et al., J. Immunol. 35:1-21 (1990); Kohlerand Milstein, Nature 256:495-497 (1975)), the trioma technique, thehuman B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72(1983); Cole et al., in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc. (1985), pp. 77-96).

Methods for immunization are well known in the art. Such methods includesubcutaneous or interperitoneal injection of the polypeptide. Oneskilled in the art will recognize that the amount of the protein encodedby the reporter gene of the present invention used for immunization willvary based on the animal which is immunized, the antigenicity of thepeptide and the site of injection.

The polypeptide or protein of the invention which is used as animmunogen may be modified or administered in an adjuvant in order toincrease the polypeptide or protein's antigenicity. Methods ofincreasing the antigenicity of a polypeptide or protein are well knownin the art and include, but are not limited to, coupling the antigenwith a heterologous protein (such as globulin or β-galactosidase) orthrough the inclusion of an adjuvant during immunization.

For monoclonal antibodies, spleen cells from the immunized animals areremoved, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, andallowed to become monoclonal antibody producing hybridoma cells.

Any one of a number of methods well known in the art can be used toidentify the hybridoma cell which produces an antibody with the desiredcharacteristics. These include screening the hybridomas with an ELISAassay, western blot analysis, or radioimmunoassay (Lutz et al., Exp.Cell Research. 175:109-124 (1988)).

Hybridomas secreting the desired antibodies are cloned and the class andsubclass is determined using procedures known in the art (Campbell, A.M., Monoclonal Antibody Technology: Laboratory Techniques inBiochemistry and Molecular Biology, Elsevier Science Publishers,Amsterdam, The Netherlands (1984)).

For polyclonal antibodies, antibody containing antisera is isolated fromthe immunized animal and is screened for the presence of antibodies withthe desired specificity using one of the above-described procedures.

The present invention further provides the above-described antibodies indetectably labeled form. Antibodies can be detectably labeled throughthe use of radioisotopes, affinity labels (such as biotin, avidin,etc.), enzymatic labels (such as horseradish peroxidase, alkalinephosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc.),paramagnetic atoms, etc. Procedures for accomplishing such labeling arewell-known in the art, for example, see (Sternberger, L. A. et al., J.Histochem. CytoChem. 18:315 (1970); Bayer, E. A. et al., Meth. Enzym.62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972); Goding, J. W.J. Immunol. Meth. 13:215 (1976)).

The labeled antibodies of the present invention can be used for invitro, in vivo, and in situ assays to identify cells or tissues in whichthe polypeptide or protein of the invention is expressed.

The present invention further provides the above-described antibodiesimmobilized on a solid support. Examples of such solid supports includeplastics such as polycarbonate, complex carbohydrates such as agaroseand sepharose, acrylic resins and such as polyacrylamide and latexbeads. Techniques for coupling antibodies to such solid supports arewell known in the art (Weir, D. M. et al., ‘Handbook of ExperimentalImmunology’ 4th Ed., Blackwell Scientific Publications, Oxford, England,Chapter 10 (1986); Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press,N.Y. (1974)). The immobilized antibodies of the present invention can beused for immuno-affinity purification of host cells that are expressingthe polypeptide or protein of the invention.

Host cells are transfected or preferably infected or transformed withthe above-described vectors, and cultured in nutrient media appropriatefor selecting transductants or transformants containing the vector.

The host cells which express the polypeptide or protein of the inventionproduct may be identified by at least four general approaches; (a)DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of genefunctions; (c) assessing the level of transcription as measured by theexpression of mRNA transcripts in the host cell; and (d) detection ofthe gene product as measured by immunoassay or by its biologicalactivity.

In the first approach, the presence of the polypeptide or protein of theinvention inserted in the vector can be detected by DNA-DNA or DNA-RNAhybridization using probes comprising nucleotide sequences that arehomologous to the polypeptide or protein of the invention, respectively,or portions or derivatives thereof.

In the second approach, the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if the polypeptide or protein of the invention is inserted within amarker gene sequence of the vector, recombinant cells containing thepolypeptide or protein of the invention can be identified by the absenceof the marker gene function. Alternatively, a marker gene can be placedin tandem with the polypeptide or protein of the invention under thecontrol of the same or different promoter used to control the expressionof the polypeptide or protein of the invention. Expression of the markerin response to induction or selection indicates expression of thepolypeptide or protein of the invention.

In the third approach, transcriptional activity of the polypeptide orprotein of the invention can be assessed by hybridization assays. Forexample, RNA can be isolated and analyzed by Northern blot using a probehomologous to the polypeptide or protein of the invention or particularportions thereof. Alternatively, total nucleic acids of the host cellmay be extracted and assayed for hybridization to such probes.

In the fourth approach, the expression of a product from the polypeptideor protein of the invention can be assessed immunologically, for exampleby Western blots, immunoassays such as radioimmuno-precipitation,enzyme-linked immunoassays and the like.

The polynucleotides of the invention also provide polynucleotidesincluding nucleotide sequences that are substantially equivalent to thepolynucleotides of the invention. Polynucleotides according to theinvention can have at least about 80%, more typically at least about90%, and even more typically at least about 95%, sequence identity to apolynucleotide of the invention. The invention also provides thecomplement of the polynucleotides including a nucleotide sequence thathas at least about 80%, more typically at least about 90%, and even moretypically at least about 95%, sequence identity to a polynucleotideencoding a polypeptide recited above. The polynucleotide can be DNA(genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithmsfor obtaining such polynucleotides are well known to those of skill inthe art and can include, for example, methods for determininghybridization conditions which can routinely isolate polynucleotides ofthe desired sequence identities.

A polynucleotide according to the invention can be joined to any of avariety of other nucleotide sequences by well-established recombinantDNA techniques (see Sambrook J et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotidesequences for joining to polypeptides include an assortment of vectors,e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and thelike, that are well known in the art. Accordingly, the invention alsoprovides a vector including a polynucleotide of the invention and a hostcell containing the polynucleotide. In general, the vector contains anorigin of replication functional in at least one organism, convenientrestriction endonuclease sites, and a selectable marker for the hostcell. Vectors according to the invention include expression vectors,replication vectors, probe generation vectors, and sequencing vectors. Ahost cell according to the invention can be a prokaryotic or eukaryoticcell and can be a unicellular organism or part of a multicellularorganism.

The sequences falling within the scope of the present invention are notlimited to the specific sequences herein described, but also include arepresentative fragment thereof, or a nucleotide sequence at least 99.9%identical to a nucleic acid of the invention. Furthermore, toaccommodate codon variability, the invention includes nucleic acidmolecules encoding the polypeptide sequences of the invention. In otherwords, in the coding region of a polypeptide sequence of the invention,substitution of one codon for another which encodes the same amino acidis expressly contemplated. Any specific sequence disclosed herein can bereadily screened for errors by resequencing a particular fragment, suchas an ORF, in both directions (i.e., sequence both strands).

The present invention further provides recombinant constructs comprisinga nucleic acid of the invention, or a fragment thereof. The recombinantconstructs of the present invention comprise a vector, such as a plasmidor viral vector, into which a nucleic acid of the invention, or afragment thereof is inserted, in a forward or reverse orientation. Inthe case of a vector comprising one of the ORFs of the presentinvention, the vector may further comprise regulatory sequences,including for example, a promoter, operably linked to the ORF. Forvectors comprising the EMFs and UMFs of the present invention, thevector may further comprise a marker sequence or heterologous ORFoperably linked to the EMF or UMF. Large numbers of suitable vectors andpromoters are known to those of skill in the art and are commerciallyavailable for generating the recombinant constructs of the presentinvention. The following vectors are provided by way of example.Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a,pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).

The isolated polynucleotide of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490(1991), in order to produce the protein or polypeptide recombinantly.Many suitable expression control sequences are known in the art. Generalmethods of expressing recombinant proteins are also known and areexemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). Asdefined herein “operably linked” means that the isolated polynucleotideof the invention and an expression control sequence are situated withina vector or cell in such a way that the protein or polypeptide isexpressed by a host cell which has been transformed (transfected) withthe ligated polynucleotide/expression control sequence.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), andtrc. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art. Generally,recombinant expression vectors will include origins of replication andselectable markers permitting transformation of the host cell, e.g., theampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and apromoter derived from a highly-expressed gene to direct transcription ofa downstream structural sequence. Such promoters can be derived fromoperons encoding glycolytic enzymes such as 3-phosphoglycerate kinase(PGK), a-factor, acid phosphatase, or heat shock proteins, among others.The heterologous structural sequence is assembled in appropriate phasewith translation initiation and termination sequences, and preferably, aleader sequence capable of directing secretion of translated protein orpolypeptide into the periplasmic space or extracellular medium.Optionally, the heterologous sequence can encode a fusion proteinincluding an N-terminal identification peptide imparting desiredcharacteristics, e.g., stabilization or simplified purification ofexpressed recombinant product. Useful expression vectors for bacterialuse are constructed by inserting a structural DNA sequence encoding adesired protein or polypeptide together with suitable translationinitiation and termination signals in operable reading phase with afunctional promoter. The vector will comprise one or more phenotypicselectable markers and an origin of replication to ensure maintenance ofthe vector and to, if desirable, provide amplification within the host.Suitable prokaryotic hosts for transformation include E. coli, Bacillussubtilis, Salmonella typhimurium and various species within the generaPseudomonas, Streptomyces, and Staphylococcus, although others may alsobe employed as a matter of choice.

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, Wis.,USA). These pBR322 ‘backbone’ sections are combined with an appropriatepromoter and the structural sequence to be expressed. Followingtransformation of a suitable host strain and growth of the host strainto an appropriate cell density, the selected promoter is induced orderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification.

Included within the scope of the nucleic acid sequences of the inventionare nucleic acid sequences that hybridize under stringent conditions toa polynucleotide of the invention, which polynucleotide is greater thanabout 10 bp, preferably 20-50 bp, and even greater than 100 bp. Inaccordance with the invention, polynucleotide sequences of theinvention, or functional equivalents thereof, may be used to generaterecombinant DNA molecules that direct the expression of thatpolynucleotide, or a functional equivalent thereof, in appropriate hostcells.

The polynucleotides of the invention are further directed to sequenceswhich encode variants of the polypeptides or proteins of the invention.These amino acid sequence variants may be prepared by methods known inthe art by introducing appropriate nucleotide changes into a native orvariant polynucleotide. There are two variables in the construction ofamino acid sequence variants: the location of the mutation and thenature of the mutation. The amino acid sequence variants of the nucleicacids are preferably constructed by mutating the polynucleotide to givean amino acid sequence that does not occur in nature. These amino acidalterations can be made at sites that differ in the nucleic acids fromdifferent species (variable positions) or in highly conserved regions(constant regions). Sites at such locations will typically be modifiedin series, e.g., by substituting first with conservative choices (e.g.,hydrophobic amino acid to a different hydrophobic amino acid) and thenwith more distant choices (e.g., hydrophobic amino acid to a chargedamino acid), and then deletions or insertions may be made at the targetsite. Amino acid sequence deletions generally range from about 1 to 30residues, preferably about 1 to 10 residues, and are typicallycontiguous. Amino acid insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one to one hundred ormore residues, as well as intrasequence insertions of single or multipleamino acid residues. Intrasequence insertions may range generally fromabout 1 to 10 amino residues, preferably from 1 to 5 residues. Examplesof terminal insertions include the heterologous signal sequencesnecessary for secretion or for intracellular targeting in different hostcells.

Amino acid sequence deletions generally range from about 1 to 30residues, preferably about 1 to 10 residues, and are typicallycontiguous. Amino acid insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one to one hundred ormore residues, as well as intrasequence insertions of single or multipleamino acid residues. Intrasequence insertions may range generally fromabout 1 to 10 amino residues, preferably from 1 to 5 residues. Examplesof terminal insertions include the heterologous signal sequencesnecessary for secretion or for intracellular targeting in different hostcells.

PCR may also be used to create amino acid sequence variants of thepolynucleotides of the invention. When small amounts of template DNA areused as starting material, primer(s) that differs slightly in sequencefrom the corresponding region in the template DNA can generate thedesired amino acid variant. PCR amplification results in a population ofproduct DNA fragments that differ from the polynucleotide templateencoding the polypeptide or protein at the position specified by theprimer. The product DNA fragments replace the corresponding region inthe plasmid and this gives the desired amino acid variant.

In a preferred method, polynucleotides encoding the polynucleotides ofthe invention are changed via site-directed mutagenesis. This methoduses oligonucleotide sequences that encode the polynucleotide sequenceof the desired amino acid variant, as well as a sufficient adjacentnucleotide on both sides of the changed amino acid to form a stableduplex on either side of the site of being changed. In general, thetechniques of site-directed mutagenesis are well known to those of skillin the art and this technique is exemplified by publications such as,Edelman et al., DNA 2:183 (1983). A versatile and efficient method forproducing site-specific changes in a polynucleotide sequence waspublished by Zoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982).PCR may also be used to create amino acid sequence variants of the novelnucleic acids. When small amounts of template DNA are used as startingmaterial, primer(s) that differs slightly in sequence from thecorresponding region in the template DNA can generate the desired aminoacid variant. PCR amplification results in a population of product DNAfragments that differ from the polynucleotide template encoding thepolypeptide at the position specified by the primer. The product DNAfragments replace the corresponding region in the plasmid and this givesthe desired amino acid variant.

A further technique for generating amino acid variants is the cassettemutagenesis technique described in Wells et al., Gene 34:315 (1985); andother mutagenesis techniques well known in the art, such as, forexample, the techniques in Sambrook et al., supra, and Current Protocolsin Molecular Biology, Ausubel et al. Due to the inherent degeneracy ofthe genetic code, other DNA sequences which encode substantially thesame or a functionally equivalent amino acid sequence may be used in thepractice of the invention for the cloning and expression of these novelnucleic acids. Such DNA sequences include those which are capable ofhybridizing to the appropriate novel nucleic acid sequence understringent conditions.

The invention encompasses polypeptides or proteins encoded by thepolynucleotides of the invention. Fragments of the polypeptides orproteins of the present invention which are capable of exhibitingbiological activity are also encompassed by the present invention.Fragments of the protein or polypeptide may be in linear form or theymay be cyclized using known methods, for example, as described in H. U.Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S.McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both ofwhich are incorporated herein by reference.

The present invention also provides both full-length and mature forms ofthe polypeptides or proteins of the invention. The full-length form ofthe such polypeptides or proteins can be identified by translation ofthe nucleotide sequence of each polynucleotide of the invention. Themature form of such polypeptide or protein may be obtained by expressionof the full-length polynucleotide in a suitable mammalian cell or otherhost cell. The sequence of the mature form of the polypeptide or proteinmay also be determinable from the amino acid sequence of the full-lengthform.

Where the protein or polypeptide of the present invention ismembrane-bound (e.g., is a receptor), the present invention alsoprovides for soluble forms of such protein or polypeptide. In such formspart or all of the intracellular and transmembrane domains of theprotein or polypeptide are deleted such that the protein or polypeptideis fully secreted from the cell in which it is expressed. Theintracellular and transmembrane domains of proteins or polypeptides ofthe invention can be identified in accordance with known techniques fordetermination of such domains from sequence information.

The invention also relates to methods for producing a polypeptide orprotein of the invention comprising growing a culture of the cells ofthe invention in a suitable culture medium, and purifying the protein orpolypeptide of the invention from the culture. For example, the methodsof the invention include a process for producing a polypeptide orprotein of the invention in which a host cell containing a suitableexpression vector that includes a polynucleotide or protein of theinvention is cultured under conditions that allow expression of theencoded polypeptide or protein. The polypeptide or protein can berecovered from the culture, conveniently from the culture medium, andfurther purified.

The invention further provides a polypeptide or protein of the inventionincluding an amino acid sequence that is substantially equivalent to anamino acid sequence encoded by a polynucleotide of the invention.Polypeptides or proteins according to the invention can have at leastabout 95%, and more typically at least about 98%, sequence identity toan amino acid sequence encoded by a polynucleotide of the invention.

The present invention further provides isolated polypeptides or proteinsencoded by the polyncueltides of the present invention or by degeneratevariants of the polynucleotides of the present invention. By ‘degeneratevariant’ is intended polynucleotides which differ from a nucleic acidfragment of the present invention (e.g., an ORF) by nucleotide sequencebut, due to the degeneracy of the genetic code, encode an identicalpolypeptide sequence. Preferred polynucleotides of the present inventionare the ORFs that encode proteins or polypeptides. A variety ofmethodologies known in the art can be utilized to obtain any one of theisolated polypeptides or proteins of the present invention. At thesimplest level, the amino acid sequence can be synthesized usingcommercially available peptide synthesizers. This is particularly usefulin producing small peptides and fragments of larger polypeptides.Fragments are useful, for example, in generating antibodies against thenative polypeptide. In an alternative method, the polypeptide or proteinis purified from bacterial cells which naturally produce the polypeptideor protein. One skilled in the art can readily follow known methods forisolating polypeptides and proteins in order to obtain one of theisolated polypeptides or proteins of the present invention. Theseinclude, but are not limited to, immunochromatography, HPLC,size-exclusion chromatography, ion-exchange chromatography, andimmuno-affinity chromatography. See, e.g., Scopes, Protein Purification:Principles and Practice, Springer-Verlag (1994); Sambrook, et al., inMolecular Cloning: A Laboratory Manual; Ausubel et al., CurrentProtocols in Molecular Biology.

The polypeptides and proteins of the present invention can alternativelybe purified from cells which have been altered to express the desiredpolypeptide or protein. As used herein, a cell is said to be altered toexpress a desired polypeptide or protein when the cell, through geneticmanipulation, is made to produce a polypeptide or protein which itnormally does not produce or which the cell normally produces at a lowerlevel. One skilled in the art can readily adapt procedures forintroducing and expressing either recombinant or synthetic sequencesinto eukaryotic or prokaryotic cells in order to generate a cell whichproduces one of the polypeptides or proteins of the present invention.The purified polypeptides or proteins can be used in in vitro bindingassays which are well known in the art to identify molecules which bindto the polypeptides or proteins. These molecules include but are notlimited to, for e.g., small molecules, molecules from combinatoriallibraries, antibodies or other proteins or polypeptides. The moleculesidentified in the binding assay are then tested for antagonist oragonist activity in in vivo tissue culture or animal models that arewell known in the art. In brief, the molecules are titrated into aplurality of cell cultures or animals and then tested for eithercell/animal death or prolonged survival of the animal/cells.

In addition, the binding molecules may be complexed with toxins, e.g.,ricin or cholera, or with other compounds that are toxic to cells. Thetoxin-binding molecule complex is then targeted to the tumor or othercell by the specificity of the binding molecule.

The protein or polypeptide of the invention may also be expressed as aproduct of transgenic animals, e.g., as a component of the milk oftransgenic cows, goats, pigs, or sheep which are characterized bysomatic or germ cells containing a polynucleotide encoding the proteinor polypeptide of the invention.

The protein or polypeptide of the invention may also be produced byknown conventional chemical synthesis. Methods for constructing theproteins or polypeptides of the present invention by synthetic means areknown to those skilled in the art. The synthetically-constructedproteins or polypeptides, by virtue of sharing primary, secondary ortertiary structural and/or conformational characteristics with proteinsor polypeptides may possess biological properties in common therewith,including protein activity. Thus, they may be employed as biologicallyactive or immunological substitutes for natural, purified proteins orpolypeptides of the invention in screening of therapeutic compounds andin immunological processes for the development of antibodies.

The proteins or polypeptides of the invention provided herein alsoinclude proteins or polypeptides characterized by amino acid sequencessimilar to those of purified proteins or polypeptides of the inventionbut into which modifications are naturally provided or deliberatelyengineered. For example, modifications in the peptide or DNA sequencescan be made by those skilled in the art using known techniques.Modifications of interest in the protein sequences may include thealteration, substitution, replacement, insertion or deletion of aselected amino acid residue in the coding sequence. For example, one ormore of the cysteine residues may be deleted or replaced with anotheramino acid to alter the conformation of the molecule. Techniques forsuch alteration, substitution, replacement, insertion or deletion arewell known to those skilled in the art (see, e.g., U.S. Pat. No.4,518,584). Preferably, such alteration, substitution, replacement,insertion or deletion retains the desired activity of the protein orpolypeptide.

Other fragments and derivatives of the polypeptides or proteins of theinvention which would be expected to retain protein activity in whole orin part and may thus be useful for screening or other immunologicalmethodologies may also be easily made by those skilled in the art giventhe disclosures herein. Such modifications are encompassed by thepresent invention.

The protein or polypeptide of the invention may also be produced byoperably linking a polynucleotide of the invention to suitable controlsequences in one or more insect expression vectors, and employing aninsect expression system. Materials and methods for baculovirus/insectcell expression systems are commercially available in kit form from,e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBat® kit), and suchmethods are well known in the art, as described in Summers and Smith,Texas Agricultural Experiment Station Bulletin No. 1555 (1987),incorporated herein by reference. As used herein, an insect cell capableof expressing a polynucleotide of the present invention is“transformed.”

The protein or polypeptide of the invention may be prepared by culturingtransformed host cells under culture conditions suitable to express therecombinant protein or polypeptide. The resulting expressed protein orpolypeptide may then be purified from such culture (i.e., from culturemedium or cell extracts) using known purification processes, such as gelfiltration and ion exchange chromatography. The purification of theprotein or polypeptide may also include an affinity column containingagents which will bind to the protein or polypeptide; one or more columnsteps over such affinity resins as concanavalin A-agarose,Heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more stepsinvolving hydrophobic interaction chromatography using such resins asphenyl ether, butyl ether, or propyl ether; or immunoaffinitychromatography.

Alternatively, the protein or polypeptide of the invention may also beexpressed in a form which will facilitate purification. For example, itmay be expressed as a fusion protein, such as those of maltose bindingprotein (MBP), glutathione-5-transferase (GST) or thioredoxin (TRX).Kits for expression and purification of such fusion proteins arecommercially available from New England BioLab (Beverly, Mass.),Pharmacia (Piscataway, N.J.) and In Vitrogen, respectively. The proteinor polypeptide can also be tagged with an epitope and subsequentlypurified by using a specific antibody directed to such epitope. One suchepitope (“Flag”) is commercially available from Kodak (New Haven,Conn.).

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the protein or polypeptide. Some or all ofthe foregoing purification steps, in various combinations, can also beemployed to provide a substantially homogeneous isolated recombinantprotein or polypeptide. The protein or polypeptide thus purified issubstantially free of other mammalian proteins and is defined inaccordance with the present invention as an “isolated protein.”

The polynucleotides and polypeptides of the present invention areexpected to exhibit one or more of the uses or biological activities(including those associated with assays cited herein) identified below.Uses or activities described for the polypeptides or proteins of thepresent invention may be provided by administration or use of suchproteins or polypeptides or by administration or use of polynucleotidesencoding such proteins or polypeptides (such as, for example, in genetherapies or vectors suitable for introduction of DNA).

The polynucleotides provided by the present invention can be used by theresearch community for various purposes. The polynucleotides can be usedto express recombinant protein or polypeptide for analysis,characterization, diagnostic or therapeutic use; as markers for tissuesin which the target protein is abnormally or normally expressed (e.g.,constitutively or at a particular stage of tissue differentiation ordevelopment or in disease states); as molecular weight markers onSouthern gels; as chromosome markers or tags (when labeled) to identifychromosomes or to map related gene positions; to compare with endogenousDNA sequences in patients to identify potential genetic disorders; asprobes to hybridize and thus discover novel, related DNA sequences; as asource of information to derive PCR primers for genetic fingerprinting;as a probe to “subtract-out” known sequences in the process ofdiscovering other novel polynucleotides; for selecting and makingoligomers for attachment to a “gene chip” or other support, includingfor examination of expression patterns; for attachment to a substrate tomake an antibody chip for examining protein (target) expression patternsor target expression levels or the presence of the target, and as anantigen to raise anti-idiotype antibodies. When the target protein bindsor potentially binds to another protein or other factor, thepolynucleotides of the invention can also be used in interaction trapassays (such as, for example, that described in Gyuris et al., Cell75:791-803 (1993)) to identify polynucleotides encoding the otherprotein or factor with which binding occurs or to identify other factorsor proteins involved in the binding interation.

The proteins or polypeptides provided by the present invention cansimilarly be used to determine biological activity, including in a panelof multiple proteins or polypeptides for high-throughput screening; as areagent (including the labeled reagent) in assays designed toquantitatively determine levels of the target protein in biologicalsamples; as markers for tissues in which the target protein of theinvention is normally or abnormally expressed (either constitutively orat a particular stage of tissue differentiation or development or in adisease state); and, of course, to isolate correlative receptors orligands. Where the target protein binds or potentially binds to anotherprotein or factor (such as, for example, in a receptor-ligandinteraction), the polypeptide of the invention can be used to identifythe other protein or factor with which binding occurs or to identifyinhibitors of the binding interaction. Proteins involved in thesebinding interactions can also be used to screen for peptide or smallmolecule inhibitors or agonists of the binding interaction.

Any or all of these research utilities are capable of being developedinto reagent grade or kit format for commercialization as researchproducts.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include withoutlimitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold SpringHarbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatiseds., 1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

Polynucleotides, proteins, and polypeptides of the present invention canalso be used as nutritional sources or supplements. Such uses includewithout limitation use as a protein or polypeptide or amino acidsupplement, use as a carbon source, use as a nitrogen source and use asa source of carbohydrate. In such cases the protein, polypeptide, orpolynucleotide of the invention can be added to the feed of a particularorganism or can be administered as a separate solid or liquidpreparation, such as in the form of powder, pills, solutions,suspensions or capsules. In the case of microorganisms, the protein,polypeptide, or polynucleotide of the invention can be added to themedium in or on which the microorganism is cultured.

A protein or polypeptide of the present invention may exhibit cytokine,cell proliferation (either inducing or inhibiting) or celldifferentiation (either inducing or inhibiting) activity or may induceproduction of other cytokines in certain cell populations, or may be anantogonist or agonist of any of the above. A polynucleotide of theinvention can encode a polypeptide exhibiting such attributes. Manyprotein factors discovered to date, including all known cytokines, haveexhibited activity in one or more factor-dependent cell proliferationassays, and hence the assays serve as a convenient confirmation ofcytokine agonist or antagonist activity. The activity of a protein orpolypeptide of the present invention is evidenced by any one of a numberof routine factor dependent cell proliferation assays for cell linesincluding, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3,MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Moleand CMK.

The activity of a protein or polypeptide of the invention may, amongother means, be measured by the following methods:

Assays for T-cell or thymocyte proliferation include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, Invitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7,Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolliet al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I.Immunol. 149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761,1994.

Assays for cytokine production and/or proliferation of spleen cells,lymph node cells or thymocytes include, without limitation, thosedescribed in: Polyclonal T cell stimulation, Kruisbeek, A. M. andShevach, E. M. In Current Protocols in Immunology. J. E. e.a. Coliganeds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; andMeasurement of mouse and human interleukin .gamma., Schreiber, R. D. InCurrent Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp.6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Measurement of Human and Murine Interleukin 2 and Interleukin 4,Bottomly, K., Davis, L. S, and Lipsky, P. E. In Current Protocols inImmunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wileyand Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211,1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse andhuman interleukin 6-Nordan, R. In Current Protocols in Immunology. J. E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto.1991; Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986;Measurement of human Interleukin 11-Bennett, F., Giannotti, J., Clark,S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. e.a.Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;Measurement of mouse and human Interleukin 9-Ciarletta, A., Giannotti,J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology.J. E. e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto.1991.

Assays for T-cell clone responses to antigens (which will identify,among others, proteins that affect APC-T cell interactions as well asdirect T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In vitro assays for Mouse LymphocyteFunction; Chapter 6, Cytokines and their cellular receptors; Chapter 7,Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad.Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun.11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takaiet al., J. Immunol. 140:508-512, 1988.

In all the above assays, the polypeptide or protein of the invention isadded into the assay system and activity of a target cytokine isdetermined in the presence and absence of the polypeptide or protein ofthe invention.

Further, the polypeptides of the invention may be used to examine theexpression level or presence of a cytokine. In alternate embodiments,the detection of a cytokine or of a the level of a cytokine will bediagnostic for a disease state or condition.

A protein or polypeptide of the present invention may also exhibitimmune stimulating or immune suppressing activity, or may be antogonistsor agonists of either activity, including without limitation theactivities for which assays are described herein. A polynucleotide ofthe invention can encode a polypeptide or protein exhibiting suchactivities. A protein or polypeptide of the invention may be useful inthe treatment and/or detection (e.g., a diagnostic) of various immunedeficiencies and disorders (including severe combined immunodeficiency(SCID)), e.g., in regulating (up or down) growth and proliferation of Tand/or B lymphocytes, as well as effecting the cytolytic activity of NKcells and other cell populations. These immune deficiencies may begenetic or be caused by viral (e.g., HIV) as well as bacterial or fungalinfections, or may result from autoimmune disorders. More specifically,infectious diseases caused by viral, bacterial, fungal or otherinfections may be treatable or detectable (e.g., a diagnostic test)using a protein or polypeptide of the present invention, includinginfections by HIV, hepatitis viruses, herpesviruses, mycobacteria,Leishmania spp., malaria spp. and various fungal infections such ascandidiasis. Of course, in this regard, a protein or polypeptide of thepresent invention may also be useful where a boost to the immune systemgenerally may be desirable, i.e., in the treatment of cancer.

Autoimmune disorders which may be treated or detected using a protein orpolypeptide of the present invention include, for example, connectivetissue disease, multiple sclerosis, systemic lupus erythematosus,rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barresyndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis,myasthenia gravis, graft-versus-host disease and autoimmune inflammatoryeye disease. Such a protein or polypeptide of the present invention mayalso to be useful in the treatment of allergic reactions and conditions,such as asthma (particularly allergic asthma) or other respiratoryproblems. Other conditions, in which immune suppression is desired(including, for example, organ transplantation), may also be treatableusing a protein or polypeptide of the present invention.

Using the proteins or polypeptides of the invention it may also bepossible to modulate immune responses, in a number of ways. Downregulation may be in the form of inhibiting or blocking an immuneresponse already in progress or may involve preventing the induction ofan immune response. The functions of activated T cells may be inhibitedby suppressing T cell responses or by inducing specific tolerance in Tcells, or both. Immunosuppression of T cell responses is generally anactive, non-antigen-specific, process which requires continuous exposureof the T cells to the suppressive agent. Tolerance, which involvesinducing non-responsiveness or anergy in T cells, is distinguishablefrom immunosuppression in that it is generally antigen-specific andpersists after exposure to the tolerizing agent has ceased.Operationally, tolerance can be demonstrated by the lack of a T cellresponse upon reexposure to specific antigen in the absence of thetolerizing agent.

Down regulating or preventing one or more antigen functions (includingwithout limitation B lymphocyte antigen functions (such as, for example,B7)), e.g., preventing high level lymphokine synthesis by activated Tcells, will be useful in situations of tissue, skin and organtransplantation and in graft-versus-host disease (GVHD). For example,blockage of T cell function should result in reduced tissue destructionin tissue transplantation. Typically, in tissue transplants, rejectionof the transplant is initiated through its recognition as foreign by Tcells, followed by an immune reaction that destroys the transplant. Theadministration of a molecule which inhibits or blocks interaction of aB7 lymphocyte antigen with its natural ligand(s) on immune cells (suchas a soluble, monomeric form of a peptide having B7-2 activity alone orin conjunction with a monomeric form of a peptide having an activity ofanother B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody),prior to transplantation can lead to the binding of the molecule to thenatural ligand(s) on the immune cells without transmitting thecorresponding costimulatory signal. Blocking B lymphocyte antigenfunction in this matter prevents cytokine synthesis by immune cells,such as T cells, and thus acts as an immunosuppressant. Moreover, thelack of costimulation may also be sufficient to anergize the T cells,thereby inducing tolerance in a subject. Induction of long-termtolerance by B lymphocyte antigen-blocking reagents may avoid thenecessity of repeated administration of these blocking reagents. Toachieve sufficient immunosuppression or tolerance in a subject, it mayalso be necessary to block the function of a combination of B lymphocyteantigens.

The efficacy of particular blocking reagents in preventing organtransplant rejection or GVHD can be assessed using animal models thatare predictive of efficacy in humans. Examples of appropriate systemswhich can be used include allogeneic cardiac grafts in rats andxenogeneic pancreatic islet cell grafts in mice, both of which have beenused to examine the immunosuppressive effects of CTLA4Ig fusion proteinsin vivo as described in Lenschow et al., Science 257:789-792 (1992) andTurka et al., Proc. Natl. Acad. Sci. USA, 89:11102-11105 (1992). Inaddition, murine models of GVHD (see Paul ed., Fundamental Immunology,Raven Press, New York, 1989, pp. 846-847) can be used to determine theeffect of blocking B lymphocyte antigen function in vivo on thedevelopment of that disease. Further, the polypeptides of the inventioncan be used to detect GVHD after organ transplant.

Blocking antigen function may also be therapeutically useful fortreating autoimmune diseases. Many autoimmune disorders are the resultof inappropriate activation of T cells that are reactive against selftissue and which promote the production of cytokines and autoantibodiesinvolved in the pathology of the diseases. Preventing the activation ofautoreactive T cells may reduce or eliminate disease symptoms.Administration of reagents which block costimulation of T cells bydisrupting receptor:ligand interactions of B lymphocyte antigens can beused to inhibit T cell activation and prevent production ofautoantibodies or T cell-derived cytokines which may be involved in thedisease process. Additionally, blocking reagents may induceantigen-specific tolerance of autoreactive T cells which could lead tolong-term relief from the disease. The efficacy of blocking reagents inpreventing or alleviating autoimmune disorders can be determined using anumber of well-characterized animal models of human autoimmune diseases.Examples include murine experimental autoimmune encephalitis, systemiclupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murineautoimmune collagen arthritis, diabetes mellitus in NOD mice and BBrats, and murine experimental myasthenia gravis (see Paul ed.,Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).Further, polypeptides of the invention can be used to diagnose an immunedisorder and/or the susceptibility of an organism for an immunedisorder.

Upregulation of an antigen function (preferably a B lymphocyte antigenfunction), as a means of up regulating immune responses, may also beuseful in therapy. Upregulation of immune responses may be in the formof enhancing an existing immune response or eliciting an initial immuneresponse. For example, enhancing an immune response through stimulatingB lymphocyte antigen function may be useful in cases of viral infection.In addition, systemic viral diseases such as influenza, the common cold,and encephalitis might be alleviated by the administration ofstimulatory forms of B lymphocyte antigens systemically.

Alternatively, anti-vital immune responses may be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro with viral antigen-pulsed APCs either expressing apeptide of the present invention or together with a stimulatory form ofa soluble peptide of the present invention and reintroducing the invitro activated T cells into the patient. Another method of enhancinganti-viral immune responses would be to isolate infected cells from apatient, transfect them with a nucleic acid encoding a protein orpolypeptide of the present invention as described herein such that thecells express all or a portion of the protein or polypeptide on theirsurface, and reintroduce the transfected cells into the patient. Theinfected cells would now be capable of delivering a costimulatory signalto, and thereby activate, T cells in vivo.

The presence of a polypeptide or protein of the present invention havingthe activity of a B lymphocyte antigen(s) on the surface of the tumorcell provides the necessary costimulation signal to T cells to induce aT cell mediated immune response against the transfected tumor cells. Inaddition, tumor cells which lack MHC class I or MHC class II molecules,or which fail to reexpress sufficient mounts of MHC class I or MHC classII molecules, can be transfected with nucleic acid encoding all or aportion of (e.g., a cytoplasmic-domain truncated portion) of an MHCclass I a chain protein and β₂ microglobulin protein or an MHC class II“chain protein and an MHC class II β chain protein to thereby expressMHC class I or MHC class II proteins on the cell surface. Expression ofthe appropriate class I or class II MHC in conjunction with a peptidehaving the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3)induces a T cell mediated immune response against the transfected tumorcell. Optionally, a gene encoding an antisense construct which blocksexpression of an MHC class II associated protein, such as the invariantchain, can also be cotransfected with a DNA encoding a peptide havingthe activity of a B lymphocyte antigen to promote presentation of tumorassociated antigens and induce tumor specific immunity. Thus, theinduction of a T cell mediated immune response in a human subject may besufficient to overcome tumor-specific tolerance in the subject.

The activity of a protein or polypeptide of the invention may, amongother means, be measured by the following methods:

Suitable assays for thymocyte or splenocyte cytotoxicity include,without limitation, those described in: Current Protocols in Immunology,Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience(Chapter 3, In vitro assays for Mouse Lymphocyte Function 3.1-3.19;Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985;Takai et al., I. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982;Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol.137:3494-3500, 1986; Bowman et al., J. Virology 61:1992-1998; Takai etal., J. Immunol. 140:508-512, 1988; Bertagnolli et al., CellularImmunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092,1994.

Assays for T-cell-dependent immunoglobulin responses and isotypeswitching (which will identify, among others, proteins that modulateT-cell dependent antibody responses and that affect Th1/Th2 profiles)include, without limitation, those described in: Maliszewski, J.Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitroantibody production, Mond, J. J. and Brunswick, M. In Current Protocolsin Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, JohnWiley and Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, amongothers, proteins that generate predominantly Th1 and CTL responses)include, without limitation, those described in: Current Protocols inImmunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.Shevach, W. Strober, Pub. Greene Publishing Associates andWiley-Interscience (Chapter 3, In vitro assays for Mouse LymphocyteFunction 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai etal., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

Dendritic cell-dependent assays (which will identify, among others,proteins expressed by dendritic cells that activate naive T-cells)include, without limitation, those described in: Guery et al., J.Immunol. 134:536-544, 1995; Inaba et al., Journal of ExperimentalMedicine 173:549-559, 1991; Macatonia et al., Journal of Immunology154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993;Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal ofExperimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal ofClinical Investigation 94:797-807, 1994; and Inaba et al., Journal ofExperimental Medicine 172:631-640, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, amongothers, proteins that prevent apoptosis after superantigen induction andproteins that regulate lymphocyte homeostasis) include, withoutlimitation, those described in: Darzynkiewicz et al., Cytometry13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca etal., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243,1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai etal., Cytometry 14:891-897, 1993; Gorczyca et al., International Journalof Oncology 1:639-648, 1992.

Assays for proteins that influence early steps of T-cell commitment anddevelopment include, without limitation, those described in: Antica etal., Blood 84:111-117, 1994; Fine et al., Cellular Immunology155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al.,Proc. Nat. Acad. Sci. USA 88:7548-7551, 1991.

A protein or polypeptide of the present invention may be useful inregulation of hematopoiesis (as an antagonist or agonist) and,consequently, in the treatment and/or detection (e.g., a diagnostic) ofmyeloid or lymphoid cell deficiencies. Even marginal biological activityin support of colony forming cells or of factor-dependent cell linesindicates involvement in regulating hematopoiesis, e.g., in supportingthe growth and proliferation of erythroid progenitor cells alone or incombination with other cytokines, thereby indicating utility, forexample, in treating and/or detecting (e.g., a diagnostic) variousanemias or for use in conjunction with irradiation/chemotherapy tostimulate the production of erythroid precursors and/or erythroid cells;in supporting the growth and proliferation of myeloid cells such asgranulocytes and monocytes/macrophages (i.e., traditional CSF activity),for example, in conjunction with chemotherapy to prevent or treatconsequent myelo-suppression; in supporting the growth and proliferationof megakaryocytes and consequently of platelets thereby allowingprevention or treatment of various platelet disorders such asthrombocytopenia, and generally for use in place of or complimentary toplatelet transfusions; and/or in supporting the growth and proliferationof hematopoietic stem cells which are capable of maturing to any and allof the above-mentioned hematopoietic cells and therefore findtherapeutic utility in various stem cell disorders (such as thoseusually treated with transplantation, including, without limitation,aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as inrepopulating the stem cell compartment post irradiation/chemotherapy,either in-vivo or ex-vivo (i.e., in conjunction with bone marrowtransplantation or with peripheral progenitor cell transplantation(homologous or heterologous)) as normal cells or genetically manipulatedfor gene therapy.

The activity of a protein or polypeptide of the invention may, amongother means, be measured by the following methods:

Suitable assays for proliferation and differentiation of varioushematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify,among others, proteins that influence embryonic differentiationhematopoiesis) include, without limitation, those described in:Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al.,Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al.,Blood 81:2903-2915, 1993.

Assays for stem cell survival and differentiation (which will identify,among others, proteins that regulate lympho-hematopoiesis) include,without limitation, those described in: Methylcellulose colony formingassays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;Primitive hematopoietic colony forming cells with high proliferativepotential, McNiece, I. K. and Briddell, R. A. In Culture ofHematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., ExperimentalHematology 22:353-359, 1994; Cobblestone area forming cell assay,Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I. Freshney, etal. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long termbone marrow cultures in the presence of stromal cells, Spooncer, E.,Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y.1994; Long term culture initiating cell assay, Sutherland, H. J. InCulture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

A protein or polypeptide of the present invention also may have utilityin compositions used for bone, cartilage, tendon, ligament and/or nervetissue growth or regeneration, as well as for wound healing and tissuerepair and replacement, and in the treatment of burns, incisions andulcers (as an antagonist or agonist).

A protein or polypeptide of the present invention, which acts as anantagonist or agonist of cartilage and/or bone growth, has applicationin the healing of bone fractures and cartilage damage or defects inhumans and other animals. Such a preparation employing a protein orpolypeptide of the invention may have prophylactic use in closed as wellas open fracture reduction and also in the improved fixation ofartificial joints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery.

A protein or polypeptide of this invention may also be used in thetreatment and/or detection (e.g., a diagnostic) of periodontal disease,and in other tooth repair processes. Such agents may provide anenvironment to attract bone-forming cells, stimulate growth ofbone-forming cells or induce differentiation of progenitors ofbone-forming cells. A protein or polypeptide of the invention may alsobe useful in the treatment of osteoporosis or osteoarthritis, such asthrough stimulation of bone and/or cartilage repair or by blockinginflammation or processes of tissue destruction (collagenase activity,osteoclast activity, etc.) mediated by inflammatory processes.

Another category of tissue regeneration activity that may beattributable to the protein or polypeptide of the present invention istendon/ligament formation. A protein or polypeptide of the presentinvention, which induces tendon/ligament-like tissue or other tissueformation in circumstances where such tissue is not normally formed, hasapplication in the healing of tendon or ligament tears, deformities andother tendon or ligament defects in humans and other animals. Such apreparation employing a tendon/ligament-like tissue inducing protein (asan antagonist or agonist) may have prophylactic use in preventing damageto tendon or ligament tissue, as well as use in the improved fixation oftendon or ligament to bone or other tissues, and in repairing defects totendon or ligament tissue. De novo tendon/ligament-like tissue formationinduced by a composition of the present invention contributes to therepair of congenital, trauma induced, or other tendon or ligamentdefects of other origin, and is also useful in cosmetic plastic surgeryfor attachment or repair of tendons or ligaments. The compositions ofthe present invention may provide environment to attract tendon- orligament-forming cells, stimulate growth of tendon- or ligament-formingcells, induce differentiation of progenitors of tendon- orligament-forming cells, or induce growth of tendon/ligament cells orprogenitors ex vivo for return in vivo to effect tissue repair. Thecompositions of the invention may also be useful in the treatment oftendinitis, carpal tunnel syndrome and other tendon or ligament defects.The compositions may also include an appropriate matrix and/orsequestering agent as a carrier as is well known in the art.

The protein or polypeptide of the present invention may also be usefulfor proliferation of neural cells and for regeneration of nerve andbrain tissue, i.e., for the treatment and/or detection (e.g., adiagnostic) of central and peripheral nervous system diseases andneuropathies, as well as mechanical and traumatic disorders, whichinvolve degeneration, death or trauma to neural cells or nerve tissue.More specifically, a protein or polypeptide may be used in the treatmentand/or detection (e.g., a diagnostic) of diseases of the peripheralnervous system, such as peripheral nerve injuries, peripheral neuropathyand localized neuropathies, and central nervous system diseases, such asAlzheimer's, Parkinson's disease, Huntington's disease, amyotrophiclateral sclerosis, and Shy-Drager syndrome. Further conditions which maybe treated in accordance with the present invention include mechanicaland traumatic disorders, such as spinal cord disorders, head trauma andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using a protein or polypeptide of the invention.

Proteins or polypeptides of the invention may also be useful to promotebetter or faster closure of non-healing wounds, including withoutlimitation pressure ulcers, ulcers associated with vascularinsufficiency, surgical and traumatic wounds, and the like.

It is expected that a protein or polypeptide of the present inventionmay also exhibit activity for generation or regeneration of othertissues, such as organs (including, for example, pancreas, liver,intestine, kidney, skin, endothelium), muscle (smooth, skeletal orcardiac) and vascular (including vascular endothelium) tissue, or forpromoting the growth of cells comprising such tissues. Part of thedesired effects may be by inhibition or modulation of fibrotic scarringto allow normal tissue to regenerate. A protein or polypeptide of theinvention may also exhibit angiogenic activity.

A protein or polypeptide of the present invention may also be useful forgut protection or regeneration and treatment of lung or liver fibrosis,reperfusion injury in various tissues, and conditions resulting fromsystemic cytokine damage.

A protein or polypeptide of the present invention may also be useful forpromoting or inhibiting differentiation of tissues described above fromprecursor tissues or cells; or for inhibiting the growth of tissuesdescribed above.

The activity of a protein or polypeptide of the invention may, amongother means, be measured by the following methods:

Assays for tissue generation activity include, without limitation, thosedescribed in: International Patent Publication No. WO95/16035 (bone,cartilage, tendon); International Patent Publication No. WO95/05846(nerve, neuronal); International Patent Publication No. WO91/07491(skin, endothelium).

Assays for wound healing activity include, without limitation, thosedescribed in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H.I. and Rovee, D. T., eds.), Year Book Medical Publishers, Inc., Chicago,as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84(1978).

A protein or polypeptide of the present invention may also exhibitagonist or antagonist activity against activin- or inhibin-relatedactivities. Inhibins are characterized by their ability to inhibit therelease of follicle stimulating hormone (FSH), while activins and arecharacterized by their ability to stimulate the release of folliclestimulating hormone (FSH). Thus, a protein or polypeptide of the presentinvention that are agonists of inhibin, may be useful as a contraceptivebased on the ability of inhibins to decrease fertility in female mammalsand decrease spermatogenesis in male mammals. Additionally, the proteinsor polypeptides of the invention that are antagonists of activin, may beuseful as a contraceptive based on the ability of activin molecules instimulating FSH release from cells of the anterior pituitary.Alternatively, the protein or polypeptide of the invention that areagonists of activin, may be useful as a fertility inducing therapeutic,based upon the ability of activin molecules in stimulating FSH releasefrom cells of the anterior pituitary. See, for example, U.S. Pat. No.4,798,885. Further, a proteins or polypeptides of the present inventionthat are antagonists of inhibin, may be useful as a fertility inducingtherapeutic, based upon the ability of inhibins to decrease fertility infemale mammals and decrease spermatogenesis in male mammals. A proteinor polypeptide of the invention may also be useful for advancement ofthe onset of fertility in sexually immature mammals, so as to increasethe lifetime reproductive performance of domestic animals such as cows,sheep and pigs.

The activity of a protein or polypeptide of the invention may, amongother means, be measured by the following methods:

Assays for activin/inhibin activity include, without limitation, thosedescribed in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al.,Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Masonet al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci.USA 83:3091-3095, 1986.

A protein or polypeptide of the present invention may be an antognist oragonist of chemotactic or chemokinetic activity (e.g., act as achemokine) for mammalian cells, including, for example, monocytes,fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelialand/or endothelial cells. Chemotactic and chemokinetic proteins can beused to mobilize or attract a desired cell population to a desired siteof action. Antagonsits or agonists of chemotactic or chemokineticproteins provide particular advantages in treatment of inflammation, orwounds and other trauma to tissues, as well as in treatment of localizedinfections. For example, attraction of lymphocytes, monocytes orneutrophils to tumors or sites of infection may result in improvedimmune responses against the tumor or infecting agent.

A protein or polypeptide or peptide is an agonist of chemotacticactivity for a particular cell population if it can stimulate, directlyor indirectly, the directed orientation or movement of such cellpopulation. vPreferably, the protein or polypeptide or peptide has theability to directly stimulate directed movement of cells. Whether aparticular protein has chemotactic activity for a population of cellscan be readily determined by employing such protein or peptide in anyknown assay for cell chemotaxis.

The activity of a protein or polypeptide of the invention may, amongother means, be measured by the following methods:

Assays for chemotactic activity (which will identify proteins thatinduce or prevent chemotaxis) consist of assays that measure the abilityof a protein to induce the migration of cells across a membrane as wellas the ability of a protein to induce the adhesion of one cellpopulation to another cell population. Suitable assays for movement andadhesion include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 6.12, Measurement of alpha and betaChemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376,1995; Lind et al. APMIS103:140-146, 1995; Muller et al Eur. J. Immunol.25:1744-1748; Gruber et al., J. of Immunol. 152:5860-5867, 1994;Johnston et al., J. of Immunol. 153:1762-1768, 1994.

A protein or polypeptide of the invention may also be an antagonist oragonist of hemostatic or thrombolytic activity. Such a protein orpolypeptide is expected to be useful in treatment and/or detection(e.g., a diagnostic) of various coagulation disorders (includinghereditary disorders, such as hemophilias) or to enhance coagulation andother hemostatic events in treating wounds resulting from trauma,surgery or other causes. A protein or polypeptide of the invention mayalso be useful for dissolving or inhibiting formation of thromboses andfor treatment and prevention of conditions resulting therefrom (such as,for example, infarction of cardiac and central nervous system vessels(e.g., stroke).

The activity of a protein or polypeptide of the invention may, amongother means, be measured by the following methods:

Assay for hemostatic and thrombolytic activity include, withoutlimitation, those described in: Linet et al., J. Clin.Pharmacol.26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987;Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins35:467-474, 1988.

A protein or polypeptide of the present invention may also demonstrateactivity as receptors, receptor ligands or antagonists or agonists ofreceptor/ligand interactions. Examples of such receptors and ligandsinclude, without limitation, cytokine receptors and their ligands,receptor kinases and their ligands, receptor phosphatases and theirligands, receptors involved in cell-cell interactions and their ligands(including without limitation, cellular adhesion molecules (such asselectins, integrins and their ligands) and receptor/ligand pairsinvolved in antigen presentation, antigen recognition and development ofcellular and humoral immune responses). Receptors and ligands are alsouseful for screening of potential peptide or small molecule inhibitorsof the relevant receptor/ligand interaction. A protein or polypeptide ofthe present invention (including, without limitation, fragments ofreceptors and ligands) may themselves be useful as inhibitors ofreceptor/ligand interactions.

The activity of a protein or polypeptide of the invention may, amongother means, be measured by the following methods:

Suitable assays for receptor-ligand activity include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28,Measurement of Cellular Adhesion under static conditions7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868,1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein etal., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol.Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.

Proteins or polypeptides of the present invention may also beantagonists or agonists of inflammation. Anti-inflammatory activity maybe achieved by providing a stimulus to cells involved in theinflammatory response, by inhibiting or promoting cell-cell interactions(such as, for example, cell adhesion), by inhibiting or promotingchemotaxis of cells involved in the inflammatory process, inhibiting orpromoting cell extravasation, or by stimulating or suppressingproduction of other factors which more directly inhibit or promote aninflammatory response. Proteins or polypeptides of the invention can beused to treat and/or detect (e.g., a diagnostic) inflammatory conditionsincluding chronic or acute conditions, including without limitationintimation associated with infection (such as septic shock, sepsis orsystemic inflammatory response syndrome (SIRS)), ischemia-reperfusioninjury, endotoxin lethality, arthritis, complement-mediated hyperacuterejection, nephritis, cytokine or chemokine-induced lung injury,inflammatory bowel disease, Crohn's disease or resulting from overproduction of cytokines such as TNF or IL-1. Proteins or polypeptides ofthe invention may also be useful to treat anaphylaxis andhypersensitivity to an antigenic substance or material.

Nervous system disorders, which can be treated and/or detected (e.g., adiagnostic) with the polypeptides or proteins of the invention includebut are not limited to nervous system injuries, and diseases ordisorders which result in either a disconnection of axons, a diminutionor degeneration of neurons, or demyelination. Nervous system lesionswhich may be treated and/or detected (e.g., a diagnostic) in a patient(including human and non-human mammalian patients) according to theinvention include but are not limited to the following lesions of eitherthe central (including spinal cord, brain) or peripheral nervoussystems:

-   -   (i) traumatic lesions, including lesions caused by physical        injury or associated with surgery, for example, lesions which        sever a portion of the nervous system, or compression injuries;    -   (ii) ischemic lesions, in which a lack of oxygen in a portion of        the nervous system results in neuronal injury or death,        including cerebral infarction or ischemia, or spinal cord        infarction or ischemia;    -   (iii) malignant lesions, in which a portion of the nervous        system is destroyed or injured by malignant tissue which is        either a nervous system associated malignancy or a malignancy        derived from non-nervous system tissue;    -   (iv) infectious lesions, in which a portion of the nervous        system is destroyed or injured as a result of infection, for        example, by an abscess or associated with infection by human        immunodeficiency virus, herpes zoster, or herpes simplex virus        or with Lyme disease, tuberculosis, syphilis;    -   (v) degenerative lesions, in which a portion of the nervous        system is destroyed or injured as a result of a degenerative        process including but not limited to degeneration associated        with Parkinson's disease, Alzheimer's disease, Huntington's        chorea, or amyotrophic lateral sclerosis;    -   (vi) lesions associated with nutritional diseases or disorders,        in which a portion of the nervous system is destroyed or injured        by a nutritional disorder or disorder of metabolism including        but not limited to, vitamin B12 deficiency, folic acid        deficiency, Wernicke disease, tobacco-alcohol amblyopia,        Marchiafava-Bignami disease (primary degeneration of the corpus        callosum), and alcoholic cerebellar degeneration;    -   (vii) neurological lesions associated with systemic diseases        including but not limited to diabetes (diabetic neuropathy,        Bell's palsy), systemic lupus erythematosus, carcinoma, or        sarcoidosis;    -   (viii) lesions caused by toxic substances including alcohol,        lead, or particular neurotoxins; and    -   (ix) demyelinated lesions in which a portion of the nervous        system is destroyed or injured by a demyelinating disease        including but not limited to multiple sclerosis, human        immunodeficiency virus-associated myelopathy, transverse        myelopathy or various etiologies, progressive multifocal        leukoencephalopathy, and central pontine myelinolysis.

Therapeutics which are useful according to the invention for treatmentof a nervous system disorder may be selected by testing for biologicalactivity in promoting the survival or differentiation of neurons. Forexample, and not by way of limitation, therapeutics which elicit any ofthe following effects may be useful according to the invention:

-   -   (i) increased survival time of neurons in culture;    -   (ii) increased sprouting of neurons in culture or in vivo;    -   (iii) increased production of a neuron-associated molecule in        culture or in vivo, e.g., choline acetyltransferase or        acetylcholinesterase with respect to motor neurons; or    -   (iv) decreased symptoms of neuron dysfunction in vivo.

Such effects may be measured by any method known in the art. Inpreferred, non-limiting embodiments, increased survival of neurons maybe measured by the method set forth in Arakawa et al. (1990, J.Neurosci. 10:3507-3515); increased sprouting of neurons may be detectedby methods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82) orBrown et al. (1981, Ann. Rev. Neurosci. 4:17-42); increased productionof neuron-associated molecules may be measured by bioassay, enzymaticassay, antibody binding, Northern blot assay, etc., depending on themolecule to be measured; and motor neuron dysfunction may be measured byassessing the physical manifestation of motor neuron disorder, e.g.,weakness, motor neuron conduction velocity, or functional disability.

In a specific embodiments, motor neuron disorders that may be treatedand/or detected (e.g., a diagnostic) according to the invention includebut are not limited to disorders such as infarction, infection, exposureto toxin, trauma, surgical damage, degenerative disease or malignancythat may affect motor neurons as well as other components of the nervoussystem, as well as disorders that selectively affect neurons such asamyotrophic lateral sclerosis, and including but not limited toprogressive spinal muscular atrophy, progressive bulbar palsy, primarylateral sclerosis, infantile and juvenile muscular atrophy, progressivebulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis andthe post polio syndrome, and Hereditary Motorsensory Neuropathy(Charcot-Marie-Tooth Disease).

A protein or polypeptide of the invention may also exhibit one or moreof the following additional activities or effects: inhibiting thegrowth, infection or function of, or killing, infectious agents,including, without limitation, bacteria, viruses, fungi and otherparasites; effecting (suppressing or enhancing) bodily characteristicsor plant characteristics, including, without limitation, height, weight,hair color, eye color, skin, fat to lean ratio or other tissuepigmentation, or organ or body part size or shape (such as, for example,breast augmentation or diminution, change in bone form or shape);effecting biorhythms or caricadic cycles or rhythms; effecting thefertility of male or female subjects; effecting the metabolism,catabolism, anabolism, processing, utilization, storage or eliminationof dietary fat, lipid, protein, carbohydrate, vitamins, minerals,co-factors or other nutritional factors or component(s); effectingbehavioral characteristics, including, without limitation, appetite,libido, stress, cognition (including cognitive disorders), depression(including depressive disorders) and violent behaviors; providinganalgesic effects or other pain reducing effects; promotingdifferentiation and growth of embryonic stem cells in lineages otherthan hematopoietic lineages; hormonal or endocrine activity; in the caseof enzymes, correcting deficiencies of the enzyme and treatingdeficiency-related diseases; treatment of hyperproliferative disorders(such as, for example, psoriasis); immunoglobulin-like activity (suchas, for example, the ability to bind antigens or complement); and theability to act as an antigen in a vaccine composition to raise an immuneresponse against such protein or another material or entity which iscross-reactive with such protein.

A protein or polypeptide of the present invention (from whatever sourcederived, including without limitation from recombinant andnon-recombinant sources) may be administered to a patient in need, byitself, or in pharmaceutical compositions where it is mixed withsuitable carriers or excipient(s) at doses to treat or ameliorate avariety of disorders. Such a composition may also contain (in additionto protein or polypeptide and a carrier) diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials well known inthe art. The term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). The characteristics ofthe carrier will depend on the route of administration. Thepharmaceutical composition of the invention may also contain cytokines,lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF,thrombopoietin, stem cell factor, and erythropoietin. The pharmaceuticalcomposition may further contain other agents which either enhance theactivity of the protein or polypeptide or compliment its activity or usein treatment. Such additional factors and/or agents may be included inthe pharmaceutical composition to produce a synergistic effect withprotein or polypeptide of the invention, or to minimize side effects.Conversely, protein or polypeptide of the present invention may beincluded in formulations of the particular cytokine, lymphokine, otherhematopoietic factor, thrombolytic or anti-thrombotic factor, oranti-inflammatory agent to minimize side effects of the cytokine,lymphokine, other hematopoietic factor, thrombolytic or anti-thromboticfactor, or anti-inflammatory agent. A protein or polypeptide of thepresent invention may be active in multimers (e.g., heterodimers orhomodimers) or complexes with itself or other proteins. As a result,pharmaceutical compositions of the invention may comprise a protein orpolypeptide of the invention in such multimeric or complexed form.

Techniques for formulation and administration of the compounds of theinstant application may be found in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition. Atherapeutically effective dose further refers to that amount of thecompound sufficient to result in amelioration of symptoms, e.g.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, a therapeutically effective dose refersto that ingredient alone. When applied to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of protein or polypeptide of thepresent invention is administered to a mammal having a condition to betreated. Protein or polypeptide of the present invention may beadministered in accordance with the method of the invention either aloneor in combination with other therapies such as treatments employingcytokines, lymphokines or other hematopoietic factors. Whenco-administered with one or more cytokines, lymphokines or otherhematopoietic factors, protein or polypeptide of the prese effectiveamount of protein or polypeptide of the present invention isadministered to a mammal having a condition to be treated. Protein orpolypeptide of the present invention may be administered in accordancewith the method of the invention either alone or in combination withother therapies such as treatments employing cytokines, lymphokines orother hematopoietic factors. When co-administered with one or morecytokines, lymphokines or other hematopoietic factors, protein orpolypeptide of the preseoutes of administration may, for example,include oral, rectal, transmucosal, or intestinal administration;parenteral delivery, including intramuscular, subcutaneous,intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections. Administration of protein or polypeptide of thepresent invention used in the pharmaceutical composition or to practicethe method of the present invention can be carried out in a variety ofconventional ways, such as oral ingestion, inhalation, topicalapplication or cutaneous, subcutaneous, intraperitoneal, parenteral orintravenous injection. Intravenous administration to the patient ispreferred.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a arthritic joints or in fibrotic tissue, often in a depot orsustained release formulation. In order to prevent the scarring processfrequently occurring as complication of glaucoma surgery, the compoundsmay be administered topically, for example, as eye drops. Furthermore,one may administer the drug in a targeted drug delivery system, forexample, in a liposome coated with a specific antibody, targeting, forexample, arthritic or fibrotic tissue. The liposomes will be targeted toand taken up selectively by the afflicted tissue.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in a conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. These pharmaceuticalcompositions may be manufactured in a manner that is itself known, e.g.,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Proper formulation is dependent upon the route ofadministration chosen. When a therapeutically effective amount ofprotein or polypeptide of the present invention is administered orally,protein or polypeptide of the present invention will be in the form of atablet, capsule, powder, solution or elixir. When administered in tabletform, the pharmaceutical composition of the invention may additionallycontain a solid carrier such as a gelatin or an adjuvant. The tablet,capsule, and powder contain from about 5 to 95% protein or polypeptideof the present invention, and preferably from about 25 to 90% protein orpolypeptide of the present invention. When administered in liquid form,a liquid carrier such as water, petroleum, oils of animal or plantorigin such as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils may be added. The liquid form of the pharmaceuticalcomposition may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% byweight of protein or polypeptide of the present invention, andpreferably from about 1 to 50% protein or polypeptide of the presentinvention.

When a therapeutically effective amount of protein or polypeptide of thepresent invention is administered by intravenous, cutaneous orsubcutaneous injection, protein or polypeptide of the present inventionwill be in the form of a pyrogen-free, parenterally acceptable aqueoussolution. The preparation of such parenterally acceptable proteinsolutions, having due regard to pH, isotonicity, stability, and thelike, is within the skill in the art. A preferred pharmaceuticalcomposition for intravenous, cutaneous, or subcutaneous injection shouldcontain, in addition to protein or polypeptide of the present invention,an isotonic vehicle such as Sodium Chloride Injection, Ringer'sInjection, Dextrose Injection, Dextrose and Sodium Chloride Injection,Lactated Ringer's Injection, or other vehicle as known in the art. Thepharmaceutical composition of the present invention may also containstabilizers, preservatives, buffers, antioxidants, or other additivesknown to those of skill in the art. For injection, the agents of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. The compounds maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. In additionto the formulations described previously, the compounds may also beformulated as a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the inventionis a cosolvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. The cosolventsystem may be the VPD co-solvent system. VPD is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5%dextrose in water solution. This co-solvent system dissolves hydrophobiccompounds well, and itself produces low toxicity upon systemicadministration. Naturally, the proportions of a co-solvent system may bevaried considerably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied: for example, other low-toxicity nonpolar surfactants maybe used instead of polysorbate 80; the fraction size of polyethyleneglycol may be varied; other biocompatible polymers may replacepolyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars orpolysaccharides may substitute for dextrose. Alternatively, otherdelivery systems for hydrophobic pharmaceutical compounds may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Certain organic solventssuch as dimethylsulfoxide also may be employed, although usually at thecost of greater toxicity. Additionally, the compounds may be deliveredusing a sustained-release system, such as semipermeable matrices ofsolid hydrophobic polymers containing the therapeutic agent. Various ofsustained-release materials have been established and are well known bythose skilled in the art. Sustained-release capsules may, depending ontheir chemical nature, release the compounds for a few weeks up to over100 days. Depending on the chemical nature and the biological stabilityof the therapeutic reagent, additional strategies for proteinstabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols. Many of the proteinase inhibitingcompounds of the invention may be provided as salts withpharmaceutically compatible counterions. Such pharmaceuticallyacceptable base addition salts are those salts which retain thebiological effectiveness and properties of the free acids and which areobtained by reaction with inorganic or organic bases such as sodiumhydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine,monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate,triethanol amine and the like.

The pharmaceutical composition of the invention may be in the form of acomplex of the protein(s) or polypeptide(s) of present invention alongwith protein or peptide antigens. The protein and/or peptide antigenwill deliver a stimulatory signal to both B and T lymphocytes. Blymphocytes will respond to antigen through their surface immunoglobulinreceptor. T lymphocytes will respond to antigen through the T cellreceptor (TCR) following presentation of the antigen by MHC proteins.MHC and structurally related proteins including those encoded by class Iand class II MHC genes on host cells will serve to present the peptideantigen(s) to T lymphocytes. The antigen components could also besupplied as purified MHC-peptide complexes alone or with co-stimulatorymolecules that can directly signal T cells. Alternatively antibodiesable to bind surface immunoglobulin and other molecules on B cells aswell as antibodies able to bind the TCR and other molecules on T cellscan be combined with the pharmaceutical composition of the invention.The pharmaceutical composition of the invention may be in the form of aliposome in which protein or polypeptide of the present invention iscombined, in addition to other pharmaceutically acceptable carriers,with amphipathic agents such as lipids which exist in aggregated form asmicelles, insoluble monolayers, liquid crystals, or lamellar layers inaqueous solution. Suitable lipids for liposomal formulation include,without limitation, monoglycerides, diglycerides, sulfatides,lysolecithin, phospholipids, saponin, bile acids, and the like.Preparation of such liposomal formulations is within the level of skillin the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871;4,501,728; 4,837,028; and 4,737,323, all of which are incorporatedherein by reference.

The amount of protein or polypeptide of the present invention in thepharmaceutical composition of the present invention will depend upon thenature and severity of the condition being treated, and on the nature ofprior treatments which the patient has undergone. Ultimately, theattending physician will decide the amount of protein or polypeptide ofthe present invention with which to treat each individual patient.Initially, the attending physician will administer low doses of proteinor polypeptide of the present invention and observe the patient'sresponse. Larger doses of protein or polypeptide of the presentinvention may be administered until the optimal therapeutic effect isobtained for the patient, and at that point the dosage is not increasedfurther. It is contemplated that the various pharmaceutical compositionsused to practice the method of the present invention should containabout 0.01 μg to about 100 mg (preferably about 0.1 μg to about 10 mg,more preferably about 0.1 μg to about 1 mg) of protein or polypeptide ofthe present invention per kg body weight. For compositions of thepresent invention which are useful for bone, cartilage, tendon orligament regeneration, the therapeutic method includes administering thecomposition topically, systematically, or locally as an implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Further, the composition may desirably be encapsulated or injectedin a viscous form for delivery to the site of bone, cartilage or tissuedamage. Topical administration may be suitable for wound healing andtissue repair. Therapeutically useful agents other than a protein orpolypeptide of the invention which may also optionally be included inthe composition as described above, may alternatively or additionally,be administered simultaneously or sequentially with the composition inthe methods of the invention. Preferably for bone and/or cartilageformation, the composition would include a matrix capable of deliveringthe protein-containing composition to the site of bone and/or cartilagedamage, providing a structure for the developing bone and cartilage andoptimally capable of being resorbed into the body. Such matrices may beformed of materials presently in use for other implanted medicalapplications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalciumphosphate. The bioceramics may be altered in composition, suchas in calcium-aluminate-phosphate and processing to alter pore size,particle size, particle shape, and biodegradability. Presently preferredis a 50:50 (mole weight) copolymer of lactic acid and glycolic acid inthe form of porous particles having diameters ranging from 150 to 800microns. In some applications, it will be useful to utilize asequestering agent, such as carboxymethyl cellulose or autologous bloodclot, to prevent the protein or polypeptide compositions fromdisassociating from the matrix.

A preferred family of sequestering agents is cellulosic materials suchas alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropyl-methylcellulose, andcarboxymethylcellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorbtion of the protein or polypeptide from thepolymer matrix and to provide appropriate handling of the composition,yet not so much that the progenitor cells are prevented frominfiltrating the matrix, thereby providing the protein or polypeptidethe opportunity to assist the osteogenic activity of the progenitorcells. In further compositions, proteins or polypeptides of theinvention may be combined with other agents beneficial to the treatmentof the bone and/or cartilage defect, wound, or tissue in question. Theseagents include various growth factors such as epidermal growth factor(EGF), platelet derived growth factor (PDGF), transforming growthfactors (TGF-.alphA. and TGF-.beta.), and insulin-like growth factor(IGF).

The therapeutic compositions are also presently valuable for veterinaryapplications. Particularly domestic animals and thoroughbred horses, inaddition to humans, are desired patients for such treatment withproteins or polypeptides of the present invention. The dosage regimen ofa protein-containing pharmaceutical composition to be used in tissueregeneration will be determined by the attending physician consideringvarious factors which modify the action of the proteins or polypeptides,e.g., amount of tissue weight desired to be formed, the site of damage,the condition of the damaged tissue, the size of a wound, type ofdamaged tissue (e.g., bone), the patient's age, sex, and diet, theseverity of any infection, time of administration and other clinicalfactors. The dosage may vary with the type of matrix used in thereconstitution and with inclusion of other proteins in thepharmaceutical composition. For example, the addition of other knowngrowth factors, such as IGF I (insulin like growth factor I), to thefinal composition, may also effect the dosage. Progress can be monitoredby periodic assessment of tissue/bone growth and/or repair, for example,X-rays, histomorphometric determinations and tetracycline labeling.

Polynucleotides of the present invention can also be used for genetherapy. Such polynucleotides can be introduced either in vivo or exvivo into cells for expression in a mammalian subject. Polynucleotidesof the invention may also be administered by other known methods forintroduction of nucleic acid into a cell or organism (including, withoutlimitation, in the form of viral vectors or naked DNA).

Cells may also be cultured ex vivo in the presence of proteins orpolypeptides of the present invention in order to proliferate or toproduce a desired effect on or activity in such cells. Treated cells canthen be introduced in vivo for therapeutic purposes.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amounts is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein. For any compound used in the methodof the invention, the therapeutically effective dose can be estimatedinitially from cell culture assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of the C-proteinase activity). Such information can be usedto more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms or a prolongation of survivalin a patient. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds which exhibit high therapeutic indicesare preferred. The data obtained from these cell culture assays andanimal studies can be used in formulating a range of dosage for use inhuman. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. See, e.g.,Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1 p. 1.Dosage amount and interval may be adjusted individually toprovide plasma levels of the active moiety which are sufficient tomaintain the C-proteinase inhibiting effects, or minimal effectiveconcentration (MEC). The MEC will vary for each compound but can beestimated from in vitro data; for example, the concentration necessaryto achieve 50-90% inhibition of the C-proteinase using the assaysdescribed herein. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. However, HPLCassays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%. In cases of local administration or selectiveuptake, the effective local concentration of the drug may not be relatedto plasma concentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may, for example, comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. Compositions comprisinga compound of the invention formulated in a compatible pharmaceuticalcarrier may also be prepared, placed in an appropriate container, andlabelled for treatment of an indicated condition.

In one application of this embodiment, a nucleotide sequence of thepresent invention can be recorded on computer readable media. As usedherein, ‘computer readable media’ refers to any medium which can be readand accessed directly by a computer. Such media include, but are notlimited to: magnetic storage media, such as floppy discs, hard discstorage medium, and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. A skilled artisan canreadily appreciate how any of the presently known computer readablemediums can be used to create a manufacture comprising computer readablemedium having recorded thereon a nucleotide sequence of the presentinvention. As used herein, ‘recorded’ refers to a process for storinginformation on computer readable medium. A skilled artisan can readilyadopt any of the presently known methods for recording information oncomputer readable medium to generate manufactures comprising thenucleotide sequence information of the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon anucleotide sequence of the present invention. The choice of the datastorage structure will generally be based on the means chosen to accessthe stored information. In addition, a variety of data processorprograms and formats can be used to store the nucleotide sequenceinformation of the present invention on computer readable medium. Thesequence information can be represented in a word processing text file,formatted in commercially-available software such as WordPerfect andMicrosoft Word, or represented in the form of an ASCII file, stored in adatabase application, such as DB2, Sybase, Oracle, or the like. Askilled artisan can readily adapt any number of dataprocessorstructuring formats (e.g. text file or database) in order to obtaincomputer readable medium having recorded thereon the nucleotide sequenceinformation of the present invention. Computer software is publiclyavailable which allows a skilled artisan to access sequence informationprovided in a computer readable medium. The examples which followdemonstrate how software which implements the BLAST (Altschul et al., J.Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem.17:203-207 (1993)) search algorithms on a Sybase system is used toidentify open reading frames (ORFs) within a nucleic acid sequence. SuchORFs may be protein or polypeptide encoding fragments and may be usefulin producing commercially important protein or polypeptides such asenzymes used in fermentation reactions and in the production ofcommercially useful metabolites.

As used herein, ‘a computer-based system’ refers to the hardware means,software means, and data storage means used to analyze the nucleotidesequence information of the present invention. The minimum hardwaremeans of the computer-based systems of the present invention comprises acentral processing unit (CPU), input means, output means, and datastorage means. A skilled artisan can readily appreciate that any one ofthe currently available computer-based systems are suitable for use inthe present invention. As stated above, the computer-based systems ofthe present invention comprise a data storage means having storedtherein a nucleotide sequence of the present invention and the necessaryhardware means and software means for supporting and implementing asearch means. As used herein, ‘data storage means’ refers to memorywhich can store nucleotide sequence information of the presentinvention, or a memory access means which can access manufactures havingrecorded thereon the nucleotide sequence information of the presentinvention.

As used herein, ‘search means’ refers to one or more programs which areimplemented on the computer-based system to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. Search means are used to identify fragments orregions of a known sequence which match a particular target sequence ortarget motif. A variety of known algorithms are disclosed publicly and avariety of commercially available software for conducting search meansare and can be used in the computer-based systems of the presentinvention. Examples of such software includes, but is not limited to,MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). A skilled artisancan readily recognize that any one of the available algorithms orimplementing software packages for conducting homology searches can beadapted for use in the present computer-based systems. As used herein, a‘target sequence’ can be any nucleic acid or amino acid sequence of sixor more nucleotides or two or more amino acids. A skilled artisan canreadily recognize that the longer a target sequence is, the less likelya target sequence will be present as a random occurrence in thedatabase. The most preferred sequence length of a target sequence isfrom about 10 to 100 amino acids or from about 30 to 300 nucleotideresidues. However, it is well recognized that searches for commerciallyimportant fragments, such as sequence fragments involved in geneexpression and protein or polypeptide processing, may be of shorterlength.

As used herein, ‘a target structural motif,’ or ‘target motif,’ refersto any rationally selected sequence or combination of sequences in whichthe sequence(s) are chosen based on a three-dimensional configurationwhich is formed upon the folding of the target motif. There are avariety of target motifs known in the art. Protein or polypeptide targetmotifs include, but are not limited to, enzyme active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, hairpin structures and inducible expression elements(protein or polypeptide binding sequences).

The present invention further provides methods to identify the presenceor expression of one of the targets recognized by a polypeptide orprotein of the present invention, or homolog thereof, in a test sample.

In general, methods for detecting a target recognized by a polypeptideor protein of the invention can comprise contacting a sample with apolypeptide or protein of the invention that binds to and forms acomplex with the target for a period sufficient to form a complex, anddetecting the complex, so that if a complex is detected, a target of theinvention is detected in the sample.

In detail, such methods comprise incubating a test sample with one ormore of the antibodies of the present invention and assaying for bindingof the antibodies to the target within the test sample.

Conditions for incubating an antibody, including a Fab fragment of theinvention, with a test sample vary. Incubation conditions depend on theformat employed in the assay, the detection methods employed, and thetype and nature of the antibody used in the assay. One skilled in theart will recognize that any one of the commonly available amplificationor immunological assay formats can readily be adapted to employ theantibodies of the present invention. Examples of such assays can befound in Chard, T., An Introduction to Radioimmunoassay and RelatedTechniques, Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, P., Practice and Theory of immunoassays: LaboratoryTechniques in Biochemistry and Molecular Biology, Elsevier SciencePublishers, Amsterdam, The Netherlands (1985); and Kuwata et al.,BioChem. Biophys. Res. Commun. 245:764-73 (1998), Hillenkamp et al.,Anal. Chem. 63:1193-202 (1991), U.S. Pat. Nos. 5,111,937 and 5,719,060.The test samples of the present invention include cells, protein orpolypeptide or membrane extracts of cells, or biological fluids such assputum, blood, serum, plasma, or urine. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing protein or polypeptideextracts or membrane extracts of cells are well known in the art and canbe readily be adapted in order to obtain a sample which is compatiblewith the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention. Specifically, the invention provides a compartment kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the antibodies of the presentinvention; and (b) one or more other containers comprising one or moreof the following: wash reagents, reagents capable of detecting presenceof a bound antibody.

In detail, a compartment kit includes any kit in which reagents arecontained in separate containers. Such containers include small glasscontainers, plastic containers or strips of plastic or paper. Suchcontainers allows one to efficiently transfer reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother. Such containers will include a container which will accept thetest sample, a container which contains the antibodies used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, etc.), and containers which contain thereagents used to detect the bound antibody or probe. Types of detectionreagents include labeled secondary antibodies, or in the alternative, ifthe primary antibody is labeled, the enzymatic, or antibody bindingreagents which are capable of reacting with the labeled antibody. Oneskilled in the art will readily recognize that the disclosed probes andantibodies of the present invention can be readily incorporated into oneof the established kit formats which are well known in the art.

Using the polypeptides or proteins of the invention, the presentinvention further provides methods of obtaining and identifying agentswhich bind to a target recognized by the polypeptide or protein. Indetail, said method comprises the steps of:

-   -   (a) contacting a target with an isolated protein or polypeptide        of the present invention; and    -   (b) determining whether the target binds to said protein or        polypeptide.

In general, such methods for identifying compounds that bind to apolypeptide of the invention can comprise contacting a compound with apolypeptide of the invention for a time sufficient to form apolypeptide/compound complex, and detecting the complex, so that if apolypeptide/compound complex is detected, a compound that binds to apolynucleotide of the invention is identified.

Methods for identifying compounds that bind to a polypeptide of theinvention can also comprise contacting a compound with a polypeptide ofthe invention in a cell for a time sufficient to form apolypeptide/compound complex, wherein the complex drives expression of areceptor gene sequence in the cell, and detecting the complex bydetecting reporter gene sequence expression, so that if apolypeptide/compound complex is detected, a compound that binds apolypeptide of the invention is identified.

Compounds identified via such methods can include compounds whichmodulate the activity of a target recognized by a polypeptide or proteinof the invention (that is, increase or decrease the target's activity,relative to activity observed in the absence of the compound).Alternatively, compounds identified via such methods can includecompounds which modulate the expression of a polynucleotide of theinvention (that is, increase or decrease expression relative toexpression levels observed in the absence of the compound). Compounds,such as compounds identified via the methods of the invention, can betested using standard assays well known to those of skill in the art fortheir ability to modulate activity/expression.

The agents screened in the above assay can be, but are not limited to,peptides, carbohydrates, vitamin derivatives, or other pharmaceuticalagents. The agents can be selected and screened at random or rationallyselected or designed using protein modeling techniques.

For random screening, agents such as peptides, carbohydrates,pharmaceutical agents and the like are selected at random and areassayed for their ability to bind to the target recognized by thepolypeptide or protein of the present invention.

Alternatively, agents may be rationally selected or designed. As usedherein, an agent is said to be ‘rationally selected or designed’ whenthe agent is chosen based on the configuration of the particular proteinor polypeptide. For example, one skilled in the art can readily adaptcurrently available procedures to generate peptides, pharmaceuticalagents and the like capable of binding to a specific peptide sequence inorder to generate rationally designed antipeptide peptides, for examplesee Hurby et al., Application of Synthetic Peptides: Antisense Peptides,‘In Synthetic Peptides, A User's Guide, W.H. Freeman, N.Y. (1992), pp.289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), orpharmaceutical agents, or the like.

In addition to the foregoing, one class of agents of the presentinvention, as broadly described, can be used to control gene expressionthrough binding to one of the ORFs or EMFs of the present invention. Asdescribed above, such agents can be randomly screened or rationallydesigned/selected. Targeting the ORF or EMF allows a skilled artisan todesign sequence specific or element specific agents, modulating theexpression of either a single ORF or multiple ORFs which rely on thesame EMF for expression control.

As choice of antibody format, we preferred the Fab format above the scFvformat, because the Fab format allows rapid high through-putaffinity-screening assays for crude antibody preparations. Many scFv'sindeed form higher molecular weight species including dimers (Weidner,et al., (1992) J. Biol. Chem. 267, 10281-10288; Holliger, et al., (1993)Proc. Natl. Acad. Sci. U.S.A. 90, 6444-6448) and trimers (Kortt et al.,(1997) Protein Eng. 10, 423-433), which complicate both selection andcharacterisation. We chose the Fab display format in which a variabledomain from a heavy or light chain gene is linked to a phage coatprotein, and in some embodiments, also carries a tag for detection andpurification. The other chain is expressed as separate fragment secretedinto the periplasm, where it can pair with the gene that is in a proteinfusion with the phage coat protein (Hoogenboom, et al., (1991) NucleicAcids Res. 19, 4133-4137). In some embodiments, the phage coat protein apIII coat protein. In other embodiments, the variable domain from aheavy chain gene is fused to the phage coat protein and the light chaingene is expressed as a separate fragment.

The choice for the Fab format was based on the notion that the monomericappearance of the Fab permits the rapid screening of large numbers ofclones for kinetics of binding (off-rate) with crude protein fractions.This reduces the time for post-selection analysis dramatically whencompared to that needed for selected single-chain Fv (scFv) antibodiesfrom phagemid libraries (Vaughan, et al., (1996) Nat. Biotechnol. 14,309-314; Sheets, et al., (1998) Proc. Natl. Acad. Sci. U.S.A. 95,6157-6162), or Fab fragments from other phage libraries (Griffiths, etal., (1993) EMBO J. 12, 725-734).

The Fab library of the invention produced on average 14 different Fab'sagainst 6 antigens that were tested. These include tetanus toxoid, thehapten phenyl-oxazolone, the breast cancer associated MUC1 antigen andthree highly related glycoprotein hormones: human Chorionic Gonadotropin(‘hCG’), human Luteinizing Hormone (‘hLH’) and human FollicleStimulating Hormone (‘hFSH’). For the glycoprotein hormones, the Fablibrary of the invention produced a panel of either homone-specific orcross-reactive antibodies. Thus, without using sophisticated selectionprotocols, hormone specific as well as cross-reactive Fab's wereretrieved against these highly homologous glycohormones, demonstratingthat the library is a rich source of antibody specificities. Theaffinities of the anti-glycohormone antibodies varied between 2.7 and 38nM. Finally, the Fab-format indeed permitted the rapid screening and areliable ranking of individual clones based on off-rate using crudefractions.

Furthermore, the specificities of the antibodies obtained by selectionson the gonadotropins are unique: due to the high degree of homologybetween hLH and hCG it has been very difficult to isolate hCG specificmonoclonal antibodies with the hybridoma technology, whereas there arevery few hLH specific antibodies (Moyle, et al., (1990) J. Biol. Chem.265, 8511-8518; Cole, (1997) Clin. Chem. 43, 2233-2243). Using astraight forward selection procedure, taking no precaution to avoid theselection of cross-reactive Fab's, we have readily isolated fragmentswith all possible specificities: Fab's specific for any of the threehormones hCG, hLH and hFSH, and cross-reactive Fab's recognizing thecommon α-chain or epitopes on the β-chain shared by hCG and hLH. Theseselections demonstrated that antibodies directed against differentepitopes within single antigen molecules can be retrieved from thelibrary. The Fab library of the invention permits the monitoring ofselections with polyclonal phage preparations and large scale screeningof antibody off-rates with unpurified Fab fragments.

Overall, antibodies with off-rates in the order of 10⁻² to 10⁻⁴ s⁻¹ andaffinities up to 2.7 nM, were recovered. The kinetics of these phageantibodies are of the same order of magnitude as antibodies associatedwith a secondary immune response.

An indication that antibodies from the Fab library behave similarly orbetter than antibodies from a scFv library with regards to affinitycomes from a comparison of selections of two different libraries on thesame two antigens under identical conditions. Antibodies to MUC1selected from a large naVve scFv library (Henderikx et al., (1998)Cancer Res. 58, 4324-4332) have faster off-rates then the equivalentFab's isolated from the library described in this study. Further, theyshow a very distinct V-gene usage and have a different fine specificity.Similarly, when comparing the off-rates of phage antibodies against thepancarcinoma marker Epithelial Glycoprotein-2, one of the Fab's selectedfrom the present library appears to have a 10-fold slower off-rate thanthe best scFv (Vaughan et al., (1996) Nat. Biotechnol. 14, 309-314).

The affinities of the selected antibody fragments is, however, very muchdependent on the antigen used for selection. Sheets and colleaguesreported an affinity varying between 26 and 71 nM for the selected scFvfragments specific for the anti-Clostridia botulinum neurotoxin type Afragments, whereas for antibodies to the extracellular domain of humanErbB-2, K_(d)'s between 0.22 and 4.03 nM were found (Sheets et al.,(1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6157-6162). The affinities ofthe gonadotropin specific Fab's selected from our library varied between2.7 and 38 nM, which is comparable to the protein binding scFv's fromthe naVve library made by Vaughan et al. and Sheets et al., andapproaches the values of the best antibodies in their kind.

The size of the Fab library of the invention is not only important foraffinity, but it also determines the success rate of selection ofantibodies against a large set of different antigens. In this respectthe Fab library of the invention performs very well: over 24 antibodiesto the hapten phOx, and on average 13 antibodies against the otherantigens were selected.

In the limited set of 14 Fab clones that were sequenced, we identifyantibodies with variable region genes from all large V-gene families,including V_(H1/3/4), V_(61/3), and V_(81/2), but also less frequentlyused segments of family V_(H6), V_(62/7) and V₈₇ were retrieved. Mostlikely the use of an extended set of variable region gene primers,designed on the most recent sequence information of the germlineV-regions, and/or the separate PCRs, combined with partially separatecloning, ensured access to a highly diverse sample of the human V-generepertoire.

According to the present invention, a library is prepared frompolynucleotides which are capable of encoding the desired specificbinding pair member. A variety of techniques exist for preparing thelibrary, which may be prepared, for example, from either genomic DNA orcDNA. See, e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989, which is incorporated herein by reference. Cells mayserve as the source of the polynucleotides which encode the specificbinding pair members of interest. Enrichment procedures and means foramplifying the regions containing the gene(s), may be employed. Forinstance, when the desired specific binding pair member is an antibody,RNA and or genomic DNA may be prepared, for example, from spleen cellsobtained from an unimmunized animal, from an animal immunized withtarget(s) of interest, from hybridoma cells, or from lymphoblastoidcells. The library of antibodies obtained from the unimmunized animalscontain an unbiased representation of the entire antibody repertoire,while the library of antibodies obtained from the immunized animalscontain a biased population of antibodies directed against epitopes ofthe target(s). Spleen cells, or immune cells from other tissues or thecirculatory system may be obtained from a variety of animal species,such as human, mouse, rat, equine, bovine, avian, etc.

Amplification of messenger RNA (mRNA) isolated from cells of interest,such as spleen or hybridoma cells, may be performed according toprotocols outlined in, e.g., U.S. Pat. No. 4,683,202, Orlandi, et al.Proc. Natl. Acad. Sci. USA 86:3833-3837 (1989), Sastry et al., Proc.Natl. Acad. Sci. USA 86:5728-5732 (1989), and Huse et al. Science246:1275-1281 (1989), Abelson, J. and Simon, M. (eds), Methods inEnzymology, combinatorial chemistry, Vol. 267, San Diego: Academic Press(1996), Kay, B. K., Winter, J., McCafferty, J. (eds), Phage Display ofpeptides and Proteins, a Laboratory Manual, San Diego: Academic Press(1996), each incorporated herein by reference. Oligonucleotide primersuseful in amplification protocols may be unique or degenerate orincorporate inosine at degenerate positions. Thus, for multi-chainimmunoglobulins, primers would be generally used for amplification ofsequences encoding the variable regions of both the heavy and lightchains. Restriction endonuclease recognition sequences may beincorporated into the primers to allow for the cloning of the amplifiedfragment into a vector in a predetermined reading frame for expression.

Expression libraries containing the amplified cDNA are typicallyprepared in a vector such as a bacteriophage or phagemid. Thecharacteristics of the suitable bacteriophage or phagemid depends on thespecific embodiment employed, and will generally be those whichconveniently allow insertion of the recombinant polynucleotides intohost cells by in vitro packaging or transformation.

Host cells are then infected with the phage or phagemid and helperphage, and cultivated under conditions allowing for the expression andassembly of phage particles. In one embodiment, the appropriate hostcells for the bacteriophage or phagemids of the invention are variousstrains of E. coli, specific examples depending on which of the severalsuitable vectors is chosen. Of course, phage or phagemid havingbacterial hosts other than E. coli may also be used.

To enrich for and isolate phage particles or phage which contain clonedlibrary sequences that encode a desired specific binding pair member,and thus to ultimately isolate the nucleic acid sequences themselves,phage particles or phage harvested from the host cells are affinitypurified. A target or binding partner for the desired specific bindingpair member is used in the affinity purification. For example, when thedesired specific binding pair member is an antibody which specificallybinds a particular target, the target is used to retrieve phageparticles or phage having the desired antibody on its outer surface. Thetarget is typically adsorbed to an insoluble substrate, such as aparticle or bead or plate. The phage particles or phage so obtained maythen be amplified by infecting into host cells (with helper phage forthe phage particles containing the phagemids). Additional rounds ofaffinity enrichment and amplification may be employed until the desiredlevel of enrichment is reached or the desired phage particles or phageare no longer enriched relative to the background phage particles orphage.

The enriched antibody-phage particles or phage are also screened withadditional detection techniques such as expression plaque (or colony)lift (see, e.g., Young and Davis, Science, 222:778-782 (1983),incorporated herein by reference) whereby the same or another bindingpartner is used as a probe. Screening may employ additional assays (fora catalytic activity, for example) which are used to detect, in situ,plaques expressing specific binding pair members having the desiredcharacteristics. The phage particles or phage obtained from thescreening protocol are infected into cells, propagated, and the phageparticle or phage DNA isolated and sequenced, and/or recloned into avector intended for gene expression in prokaryotes or eukaryotes toobtain larger amounts of the selected specific binding pair member.

In another embodiment, the specific binding pair member encoded in thelibrary (or multiple chains comprising said specific binding pairmember) is transported to an extra-cytoplasmic compartment of the hostcell, usually the periplasmic space, to facilitate processing and/orproper assembly. When extra-cytoplasmic transport of the desiredspecific binding pair member is employed, the sequences encoding thespecific binding pair member are cloned adjacent to appropriatetranscriptional and translational signals and signal peptide leadersthat will direct the mature chains to the periplasm. As above, at leastone of the chains is cloned as a fusion protein with a phage coatprotein so that the phage coat protein does not substantially interferewith the ability of the specific binding pair member of interest to binda target which is used in the affinity enrichment protocol.

A preferred example of this embodiment is the placement of a specificbinding pair member in the N-terminus region of the minor coat proteinpIII of bacteriophage fd. Before incorporation into the phage, pIIIresides in the inner membrane of the host cell with its N-terminusprotruding into the periplasm. In this configuration the polypeptide ofa specific binding pair member in the N-terminus of pIII is availablefor binding to other polypeptide chains that make up the specificbinding pair member of interest. This complex is then incorporated intothe mature phage particle or phage as it exits the cell and theC-terminus embeds in the coat of the phage particle or phage.

In this embodiment the synthesis and amplification of polynucleotides isas described above, and then is cloned into or near a vector sequenceencoding a coat protein, where the vector is, or is derived from, afilamentous phage, such as f1, fd, Pf1, M13, etc. In a preferredembodiment the filamentous phage is fd-tet. The phage vector is chosento contain a cloning site located in the 5′ region of a gene encoding aphage coat protein, such as, for example, the pIII coat protein. Anappropriate vector (e.g., fd-tet B1 which is described below) allowsoriented cloning of foreign sequences so that they are expressed at ornear the N-terminus of the mature coat protein.

A library is constructed by cloning the polynucleotides (e.g., the V_(H)region) from the donor cells into a coat protein gene (e.g., gene III,“gIII”) cloning site. The cloned sequences of, for example, the V_(H)domains are ultimately expressed as polypeptides or proteins fused tothe N-terminus of the mature coat protein on the outer, accessiblesurface of the assembled phage particles or phage.

When the desired protein is a multi-chain protein, such as an antibodyor binding fragment thereof, the polynucleotide encoding the chain(s)not cloned into a phage coat protein may be cloned directly into anappropriate site (as described below) of the vector containing the firstchain-coat protein library; or, preferably, the subsequent chain(s) maybe cloned as a separate library in a different plasmid vector,amplified, and subsequently the fragments installed in the firstchain-coat protein library vector. For example, when the first chain isan antibody heavy chain or binding fragment thereof, the ultimatedestination of light chain V_(L) cDNA sequence is in a vector thatalready contains a V_(H) sequence in a coat protein gene, thus randomlyrecombining V_(H) and V_(L) sequences in a single vector.

The second or subsequent chain of the desired multi-chain protein, suchas V_(L), is cloned so that it is expressed with a signal peptide leadersequence that will direct its secretion into the periplasm of the hostcell. For example, several leader sequences have been shown to directthe secretion of antibody sequences in E. coli, such as OmpA (Hsiung, etal., Biotechnology 4:991-995 (1986)), pelB (Better, et al., Science240:1041-1043 (1988)), phoA (Skerra and Pluckthun, Science 240:1038-1043(1988)), beta-lactamase (Zemel-Dreasen and Zamir, Gene 27:315-322(1984)), and those described in Abelson, J. and Simon, M. (eds), Methodsin Enzymology, combinatorial chemistry, Vol. 267, San Diego: AcademicPress (1996), and Kay, B. K., Winter, J., McCafferty, J. (eds), PhageDisplay of peptides and Proteins, a Laboratory Manual, San DiegoAcademic Press (1996), each incorporated herein by reference.

Generally, the successful cloning strategy utilizing a phage coatprotein, such as pIII of filamentous phage fd, will provide: (1)expression of a protein chain (or a first polypeptide chain when thedesired protein is multichained, e.g., the V_(H) chain) fused to theN-terminus of a full sized (or nearly full sized) coat protein (e.g.,pIII) and transport to the inner membrane of the host where thehydrophobic domain in the C-terminal region of the coat protein anchorsthe fusion protein in the membrane, with the N-terminus containing thechain protruding into the periplasmic space and available forinteraction with a second or subsequent chain (e.g., V_(L) to form anFab fragment) which is thus attached to the coat protein; and (2)adequate expression of a second or subsequent polypeptide chain ifpresent (e.g., V_(L)) and transport of this chain to the solublecompartment of the periplasm.

In one embodiment for affinity enrichment of desired clones, about 10³to 10⁴ library equivalents (a library equivalent is one of eachrecombinant—10⁴ equivalents of a library of 10⁹ members is 10⁹×10⁴=10¹³phage particles or phage) are incubated with target to which the desiredspecific binding pair member (e.g., antibody) is sought. The target isin one of several forms appropriate for affinity enrichment schemes. Inone example the target is immobilized on a surface or particle,optionally anchored by a tether of enough length (3 to 12 carbons, forexample) to hold the target far enough away from the surface to permitfree interaction with the antibody combining site. The library of phageparticle or phage bearing antibodies is then panned on the immobilizedtarget generally according to procedures well-known in the art, forexample, those described in Abelson, J. and Simon, M. (eds), Methods inEnzymology, combinatorial chemistry, Vol. 267, San Diego: Academic Press(1996), Kay, B. K., Winter, J., McCafferty, J. (eds), Phage Display ofpeptides and Proteins, a Laboratory Manual, San Diego: Academic Press(1996), each incorporated herein by reference.

A second example of target presentation is target attached to arecognizable ligand (again optionally with a tether of some length). Aspecific example of such a ligand is biotin. The target, so modified, isincubated with the library of phage particles or phage and bindingoccurs with both reactants in solution. The resulting complexes are thenbound to streptavidin (or avidin) through the biotin moiety. Thestreptavidin may be immobilized on a surface such as a plastic plate oron particles, in which case the complexes are physically retained; orthe streptavidin may be labelled, with a fluorophore, for example, totag the active phage/antibody for detection and/or isolation by sortingprocedures, e.g., on a fluorescence-activated cell sorter.

In one embodiment, the phage particles or phage bearing antibodieswithout the desired specificity are removed by various means, forexample, by washing. The degree and stringency of washing required willbe determined for each specific binding pair member of interest. Acertain degree of control can be exerted over the bindingcharacteristics of the antibodies recovered by adjusting the conditionsof the binding incubation and the subsequent washing. The temperature,pH, ionic strength, divalent cations concentration, and the volume andduration of the washing will select for antibodies within particularranges of affinity for the hapten. Selection based on slow dissociationrate, which is usually predictive of high affinity, is the mostpractical route. This may be done either by continued incubation in thepresence of a saturating amount of free hapten, or by increasing thevolume, number, and length of the washes. In each case, the rebinding ofdissociated antibody-phage is prevented, and with increasing time,antibody-phage of higher and higher affinity are recovered.

Antibodies with certain catalytic activities may be enriched in groupsof antibodies with high affinity for reactants (substrates andintermediates) but low affinity for products. A double screen to enrichfor antibodies with these characteristics may be useful in findingantibodies to catalyze certain reactions. Further, catalytic antibodiescapable of certain cleavage reactions may also be selected. One categoryof such reactions is the cleavage of a specific end group from amolecule. For example, a catalytic antibody to cleave a specific aminoacid from an end of a peptide may be selected by immobilizing thepeptide and panning the antibody library under conditions expected topromote binding but not cleavage (e.g., low temperature, particularionic strength, pH, cation concentration, etc., depending on the natureof the end group and the cleavage reaction) and followed by a wash. Thisallows antibodies that recognize the end group to bind and becomeimmobilized, and from this group will come those capable of cleavage. Tofind those capable of cleavage, the conditions are shifted to thosefavorable for cleavage. This step will release those antibody-phagecapable of cleaving themselves free of the immobilized peptide.

An alternative way to accomplish this is to pan for antibodies that bindto the specific end group by attaching that end group to a bonddifferent from that to be cleaved (a non-peptide bond, for example). Bysubsequent panning (of the positive phage from the first screen) on theend group attached via the proper bond under cleavage conditions, thenon-binding fraction will be enriched for those with the desiredcatalytic activity.

To elute the active antibody-phage particle or phage from theimmobilized target, after washing at the appropriate stringency, thebound (active) phage particle or phage can be recovered by eluting withpH shift. For example, pH2 or pH11 may be used, which is thenneutralized and the eluted phage are amplified by infecting ortransforming the host cells. The cells are then grown as tetracyclineresistant colonies. The colonies are scraped up and the extruded phageare purified by standard procedures as before. These phage are then usedin another round of affinity enrichment (panning), and this cycle isrepeated until the desired level of enrichment is reached or until thetarget phage are no longer enriched relative to the background phageparticles or phage. To isolate individual clones, phage particles orphage from the final round of panning and elution are infected intocells or their DNA is transformed into cells and grown on agar (usuallyL-agar) and antibiotics (usually tet) to form well separated individualcolonies, each of which is a clone carrying vectors with both V_(H) andV_(L) sequences. The single stranded DNA from phage particles or phageextruded from each colony may be isolated and DNA coding for the V_(H)and V_(L) fragments sequenced. The replicative form of the phage DNA(double stranded) may be isolated by standard means and the DNA in thecloning sites (V_(H) and V_(L) sequences) recloned into a vectordesigned for gene product expression in prokaryotes or eukaryotes toobtain larger amounts of the particular antibodies selected in thescreening process.

Phage identified as having an antibody recognized by the target ligandare propagated as appropriate for the particular phage vector used. Forfd-tet this is done in a liquid culture of rich medium (L-broth, forexample) with antibiotic (Tet) selection. The phage are harvested andDNA prepared and sequenced by standard methods to determine the DNA andamino acid sequence of the particular antibody.

The DNA may be recloned in a suitable eukaryotic or prokaryoticexpression vector and transfected into an appropriate host forproduction of large amounts of protein. Antibody is purified from theexpression system using standard procedures. The binding affinity of theantibody is confirmed by well known immunoassays with the target antigenor catalytic activity as described in Harlow and Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor, N.Y. (1988), Abelson, J. andSimon, M. (eds), Methods in Enzymology, combinatorial chemistry, Vol.267, San Diego: Academic Press (1996), Kay, B. K., Winter, J.,McCafferty, J. (eds), Phage Display of peptides and Proteins, aLaboratory Manual, San Diego: Academic Press (1996), each incorporatedherein by reference.

In another embodiment, phage particles or phage displaying the desiredspecific binding pair member are affinity purified as follows:approximately 10³-10⁴ library equivalents of phage particles or phageare reacted overnight with 1 micropgram purified antibody at 4° C. Themixture is panned by a procedure as follows. A polystyrene petri plateis coated with 1 ml of streptavidin solution (1 mg/ml in 0.1M NaHCO₃, pH8.6, 0.02% NaN₃) and is incubated overnight at 4° C. The following daythe streptavidin solution is removed. The plate is filled with 10 mlblocking solution (30 mg/ml BSA, 3 micrograms/ml streptavidin in 0.1MNaHCO₃, pH 9.2, 0.02% NaN₃) and incubated for 2 hours at roomtemperature. Two micrograms of biotinylated goat anti-mouse IgG (BRL)are added to the antibody-reacted library and incubated for 2 hours at4° C. Immediately before panning, blocking solution is removed fromstreptavidin coated plate, and the plate is washed 3 times withTBS/0.05% Tween 20. The antibody-reacted library is then added to theplate and incubated for 30 minutes at room temperature. Streptavidincoated agarose beads (BRL) may also be used for this affinitypurification. The library solution is removed and the plate is washedten times with TBS/0.05% Tween 20 over a period of 60 minutes. Boundphage are removed by adding elution buffer (1 mg/ml BSA, 0.1N HCl, pHadjusted to 2.2 with glycine) to the petri plate and incubating for 10minutes to dissociate the immune complexes. The eluate is removed,neutralized with 2M Tris (pH unadjusted) and used to infect log phaseF′-containing bacterial cells. T hese cells are then plated on LB agarplates containing tetracycline (20 .mu.g/ml), and grown overnight at 37°C. Phage—particles or phage are isolated from these plates as describedand the affinity purification process was repeated for two to threerounds. After the final round of purification, a portion of the eluateis used to infect cells and plated at low density on LB tetracyclineplates. Individual colonies are transferred to culture tubes containing2 ml LB tetracycline and grown to saturation. Phage or phagemid DNA isisolated using a method designed for the Beckman Biomek Workstation(Mardis and Roe., Biotechniques, 7:840-850 (1989)) which employs 96-wellmicrotiter plates. Single stranded DNA is sequenced by the dideoxymethod using Sequenase (U.S. Biochemicals) and an oligonucleotidesequencing primer (5′-CGATCTAAAGTTTTGTCGTCT-3′ SEQ ID NO: 2) which iscomplementary to the sequence located 40 nucleotides 3′ of the secondBstXI site in fdTetB1.

We considered a number of variables to address in the construction of anovel, very large antibody phage library: (i) the primer design wasoptimised for amplification of variable gene pools to maintain maximumdiversity; (ii) a highly efficient two-step cloning method was developedto obtain a very large naive library; (iii) an antibody format andcompatible cloning vector were chosen, which should permit the rapiddown-stream analysis of selected clones.

In order to achieve access to as many different human heavy and lightchain V-region gene segments as possible, a new set of oligonucleotideprimers was developed (Table I), the design of which was based on themost recent sequence information provided by the V-base.

The primers were designed to be the or several consensus sequences whichwould have at least a 70% homology to the respective 5′ or 3′ end basedcoding region in the human germ line gene segments of the specific Vgene family they would have to amplify. The primers would amplify atleast one V-gene segment using the PCR conditions described below, andin one embodiment are appended with appropriate positioned restrictionsites for cloning into the vector for Fab expression.

The primers should allow efficient amplification of all commonly usedV-gene segments. Further, to obtain the large sized Fab libraries of theinvention (over 10¹⁰ in diversity), we used a two-step cloningprocedure: heavy and light chain variable genes were first separatelycloned as digested PCR products, and were then combined by restrictionfragment cloning to form a large library of Fab fragments. This cloningprocedure should be a more efficient route for library construction thanthe relatively inefficient direct cloning of digested PCR-products,while avoiding the DNA instability often associated with in vivorecombination systems (Griffiths, et al., (1994) EMBO J. 13, 3245-3260).

A new phagemid vector, pCES1 (FIG. 1), was constructed, that allows thestepwise cloning of antibody fragments in Fab format. In this vectorsystem, the variable heavy chain region genes are cloned as V_(H)-genefragments; the vector supplies all Fab's with a human gamma-1 C_(H1)domain. The V_(H)C_(H1) formed by insertion of the V_(H)-gene fragmentsto the vector is fused (in the vector) to two tags for purification anddetection (a histidine tail for Immobilised Metal AffinityChromatography (Hochuli, et al., (1988) BioTechnology 6, 1321-1325) anda c-myc-derived tag (Munro, et al., H. R. (1986) Cell 46, 291-300)),followed by an amber stop codon (Hoogenboom, et al., (1991) NucleicAcids Res. 19, 4133-4137) and the minor coat protein III of filamentousphage f_(d). The antibody light chain is cloned as full V_(L)C_(L)fragment, for directed secretion and assembly with the V_(H)C_(H1) onthe phage particle.

In one embodiment, the vector comprises an expression cassette with abicistronic or double cistronic expression cassette to allow linked (forthe bicistronic) or independent (for the double cistronic) expression ofthe antibody light and heavy chain or their fusions, such expressioncassette consisting of the following elements: (1) a promoter suited fornon-inducible and inducible expression (e.g lacZ); (2) a ribosomebinding site and signal sequence preceeding the light and heavy chaincloning regions; (3) possible, but not necessarily, a region followingthe heavy or light chain cloning region that encodes a tag sequence suchas a stretch of 5-6 hsitidines or a sequence recognised by an antibodyand an amber codon; (4) a phage coat protein encoded as a fusion to the3′ end of either the heavy or light chain.

This new phage library will be a valuable source of antibodies toessentially any target. The antibodies may be used as research reagentsor as starting point for the development of therapeutic antibodies oragricultural products. As the list of sequenced genomes anddisease-related gene products is expanding rapidly, there will be agrowing need for an in vitro and eventually automated method forantibody isolation. As antibodies have been and will be ideal probes forinvestigating the nature, localization and purification of novel geneproducts, this library is envisaged to play an important role in targetvalidation and target discovery in the area of functional genomics.

Protein variants expressed on the surface of bacteriophage have beenselected on the basis of their affinity for ligand (antigen) usingchromatography, panning or adsorption to cells. Elution from affinitymatrices has been achieved by specific elution using the ligand (antigenor a related compound) or non-specific elution using, for example, 100mM triethylamine. Washing procedures remove non-specifically boundphage. The phage binds to and is eluted from the matrix according to theaffinity or the nature of the binding interaction. Specifically elutedphage are then used to infect male E. coli cells expressing the F pilus,allowing recovery of phage containing DNA encoding proteins with thedesired binding characteristics.

Selection can be made not only on the basis of specificity, but also onthe basis of affinity. Separation is readily attainable by affinitychromatography between phage expressing an antibody with a dissociationconstant of 10⁻⁸ M and one with a dissociation constant of 10⁻⁵ M.Clackson, T. et al. (1991). Nature 352: 624-628. The isolation of thelatter antibody from an immune repertoire demonstrates that antibodieswith affinities characteristic of the primary immune response can beisolated using phage technology.

Antibodies directed against cell surface antigens can also be isolatedby selective adsorption of phage on the surface of cells. Similarly, itmay be possible to incorporate negative selection with cells to removeundesired cross-reactivities with cell surface markers. As these arerather difficult and as yet poorly understood methods, methods based onthe selection on purified antigen should be used whenever possible.

Any selection for binders within a population will automatically tend toselect for high affinity variants at the expense of the lower, enrichingthe high affinity population. This has been used to good effect recentlyin the isolation of high affinity human antibodies from a naiverepertoire. Marks, J. D. et al. (1991) J. Mol. Biol. 222, 581-597. Foroptimal selection, the antigen concentration should be less than theaffinity constant. This should be borne in mind when isolating anantibody with pre-defined characteristics. Further details on variousselection methods is given in the reviews in this manual.

With such large panels of antibodies isolated, it is useful to havemethods available to readily determine the kinetic parameters of eachindividual antibody-antigen interaction. We have shown that it isfeasible to rapidly and accurately determine the off-rate ofnon-purified antibodies in periplasmic fractions prepared from smallscale cultures using surface plasmon resonance. Using this method, aseries of tetanus toxoid specific Fab's showed a monophasicdissociation, which is expected for a truly monomeric Fab-fragmentbinding to a low density antigen surface. Using this off-rate screeningassay, we determined the off-rates for the best tetanus toxoid and MUC1specific Fab's to be in the order of 10⁻² to 10⁻⁴ s⁻¹.

We tested the integrity of selected Fab's obtained from periplasmicfractions using western blots. When incubated in non-reducing samplebuffer, two products were detected with the 9E10 antibody, whichrecognises the myc-tag at the end of the CH1 domain, the major productis the intact Fab-molecule, in which an intermolecular disulfide bridgecovalently links heavy and light chain fragments; the low molecularproduct is most likely derived from non disulfide bridge linked heavychains. Analysis with anti-light chain sera reveals a similar patternand shows that the clones use a nearly equal percentage of kappa andlambda chains (found in six and seven clones respectively of a total of13 tested). Upon reduction of purified, functional antigen-binding Fabs,equal amounts of heavy and light chain are seen, while undernon-reducing conditions, the main product is represented by thedisulphide linked Fab-molecule, with an equal amount of thenon-covalently linked V_(H)C_(H1) and V_(L)C_(L) products visible.Production yields of selected hormone specific Fab's varied between 160μg and 1.43 mg Fab per litre culture, which was in the same range as wasfound for the unselected Fabs.

A panel of 14 antigen-specific Fab's was fully sequenced (3 anti-MUC1antibodies; 11 anti-gonadotropin antibodies). The heavy chain genes arederived from the four largest V_(H) families (V_(H1), V_(H3), V_(H4) andV_(H6)); the V_(L) genes belong to one of four V_(K)-families or one ofthree V_(λ)-families. Chain promiscuity is seen for the α-chain specificclone SC#4G, the α/β-LH specific clones LH#2H and LH#3G, and β-FSHspecific clone FS#8B, which all used a highly homologous V_(K2) lightchain gene segment combined with different heavy chain fragments. The 3anti-MUC1 antibodies use heavy and light chain genes derived from 2different VH and V6 families; clone MUC#9 uses a VH with a cross-over of2 segments.

The present invention is further illustrated in the following examples.Upon consideration of the present disclosure, one of skill in the artwill appreciate that many other embodiments and variations may be madein the scope of the present invention. Accordingly, it is intended thatthe broader aspects of the present invention not be limited to thedisclosure of the following examples.

As source of lymphoid tissues we used peripheral blood lymphocytes from4 healthy donors and part of a tumor-free spleen removed from a patientwith gastric carcinoma. B lymphocytes were isolated from 2-L of blood ona Ficoll-Pacque gradient. For RNA isolation, the cell pellet wasimmediately dissolved in 50 ml 8 M guanidinium thiocyanate/0.1 M2-mercaptoethanol (Chirgwin, et al., (1979) Biochemistry 18, 5294-5299).Chromosomal DNA was sheared to completion by passing through a narrowsyringe (1.2/0.5 mm gauge), and insoluble debris was removed by lowspeed centrifugation (15 min 2,934×g at room temperature). RNA waspelleted by centrifugation through a CsCl-block gradient (12 mlsupernatant on a layer of 3.5 ml 5.7 M CsCl/0.1 M EDTA; in total 4tubes) during 20 h at 125,000×g at 20° C. in a SW41-rotor (Beckman). Theyield of total RNA was approx. 600 μg. RNA was stored at −20^(E)C inethanol.

From the spleen, 2 g of tissue was used for homogenisation with apolytron in 20 ml 8 M guanidinium thiocyanate/0.1 M 2-mercaptoethanol.The total volume was increased to 80 ml with guanidinium thiocyanatebuffer, and after passage through a narrow syringe for shearing andremoval of debris, RNA was pelleted as described before, except for 15 hat 85,000×g at 20EC in a SW28.1 rotor (12 ml supernatant on 3.5 ml 5.7 MCsCl/0.1 M EDTA in 5 SW28.1 tubes). From 2 g of tissue, 3 mg of totalRNA was extracted.

Random primed cDNA was prepared with 250 μg PBL RNA, while in a separatereaction 300 μg spleen RNA was used as template. RNA was heat denaturedfor 5 min at 65° C. in the presence of 20 μg random primer (Promega),subsequently buffer and DTT were added according to the suppliersinstructions (Gibco-BRL), as well as 250 μM dNTP (Pharmacia), 800 URNAsin (40 U/μl; Promega) and 2,000 U MMLV-RT (200 U/μl; Gibco-BRL) in atotal volume of 500 μl. After 2 h at 42° C., the incubation was stoppedby a phenol/chloroform extraction; cDNA was precipitated and dissolvedin 85 μl water.

Oligonucleotides used for PCR amplification of human heavy and lightchain V-regions are described in FIG. 2. IgM-derived heavy chainvariable regions were obtained by a primary PCR with an IgM constantregion primer. All primary PCRs were carried out with separate BACKprimers and combined FOR primers, to maintain maximal diversity. ThePCR-products were reamplified with a combination of JHFOR-primers,annealing to the 3′ end of V_(H), and Sfi-tagged VHBACK-primers,annealing to the 5′ end, and subsequently cloned as V_(H)-fragments. Thelight chain V-genes of the kappa and lambda families were obtained byPCR with a set of CKFOR- or CAFOR-primer annealing to the 3′ end of theconstant domain and BACK-primers, priming at the 5′ end of theV-regions. The DNA-segments were reamplified with primers tagged withrestriction sites and cloned as V_(K)C_(K)- and V_(λ)C_(λ)-fragments.

PCR was performed in a volume of 50 μl using AmpliTaq polymerase (Cetus)and 500 pM of each primer for 28 cycles (1 min at 94^(E)C, 1 min at55^(E)C and 2 min at 72^(E)C), 9 separate IgM derived VH-amplificationswere generated with 2 μl random primed cDNA (equivalent to 6 μg PBL RNAor to 7 μg spleen RNA) as template for each reaction. For the lightchain families, 6 different V_(K)C_(K)-products and 11V_(λ)C_(λ)-products (C_(λ2)- and C_(λ7)-primers combined in eachreaction) were obtained. All products were purified from agarose gelwith the QIAex-II extraction kit (Qiagen). As input for reamplificationto introduce restriction sites, 100-200 ng purified DNA-fragment wasused as template in a 100 μl reaction volume. The large amount of input,ensuring the maintenance of variability, was checked by analysis of 4 λlof the “unamplified” PCR-mixture on agarose gel.

For the construction of the primary heavy chain and the two primarylight chain repertoires, the PCR-products, appended with restrictionsites, were gel purified prior to digestion and the different V_(H)-,V_(K)- and V_(λ)-families combined into three groups. The V_(K)C_(K)-and V_(λ)C_(λ)-fragments were digested with ApaLI and AscI, and clonedinto the phagemid vector pCES1. The V_(H)-fragments, 1.5 μg in total,were digested with SfiI and BstEII and ligated in a 100-200 μl reactionmixture with 9 U T₄-DNA ligase at room temperature to 4 μg, gel-purifiedvector pUC119-CES1 (similar to vector pCES1, but with the pIII genedeleted). The desalted ligation mixture for light or heavy chain poolswas used for electroporation of the E. coli strain TG1, to create theone-chain libraries.

The Fab library was obtained by cloning of V_(H) fragments, digestedfrom plasmid-DNA prepared from the heavy chain repertoires, into theplasmid collection containing the light chain repertoires. Plasmid DNAisolated from at least 3×10⁹ bacteria of the V_(H) library was digestedwith SfiI and BstEII for cloning in the vector that already contained λand K light chain libraries. To retain clones with internal BstEII sitein the Vλ_ (this site is relatively frequent in some λ germlineV-segments (Persic, et al., (1997) Gene 187, 9-18), and also in theconstant domain of one of the λ families), the cloning of V_(H)C_(H1) inthe λ light chain repertoire containing vector was also carried outusing SfiI and NotI cloning sites, to create a less restriction-biasedV_(λ) _(_)libary.

The rescue of phagemid particles with helper phage M13-KO7 was performedaccording to (Marks, et al., (1991) J. Mol. Biol. 222, 581-597) on 10-Lscale, using representative numbers of bacteria from the library forinoculation, to ensure the presence of at least 10 bacteria from eachclone in the start inoculum. For selections, 10¹³ cfu's (colony formingunits) were used with antigens immobilised in immunotubes (Maxisorptubes, Nunc) (Marks, et al., (1991) J. Mol. Biol. 222, 581-597) or withsoluble biotinylated antigens (Hawkins, et al., (1992b) J. Mol. Biol.226, 889-896). The amount of the immobilised antigens tetanus toxoid andthe hapten phenyl-oxazolone (conjugated to BSA in a ratio of 17 to 1)was reduced 10-fold during subsequent selection rounds, starting at 100μg/ml at round 1. Capture with biotinylated antigen in solution was usedfor a 100-mer peptide encoding five copies of the tandem repeat of MUC1(Henderikx, et al., (1998) Cancer Res. 58, 4324-4332), or with humanChorionic Gonadotropin (hCG), human Luteinizing Hormone (hLH), humanFollicle Stimulating Hormone (hFSH) and its chimeric derivative(hFSH-CTP, containing the carboxy terminal peptide from the hCGβ-subunit fused to the β-subunit of hFSH). Antigens were biotinylated ata ratio of ten to twenty molecules NHS-Biotin (Pierce) per moleculeantigen according to the suppliers recommendations. Unless statedotherwise, the antigens were used for selection at concentrations of 100nM, 30 nM and 10 nM during round 1, 2 and 3 respectively. For hFSH-CTP50, 15 and 10 nM was used respectively; for MUC1 peptide, 500, 100, 20and 5 nM was used.

Soluble Fab was produced from individual clones as described before(Marks, et al., (1991) J. Mol. Biol. 222, 581-597). Culture supernatantswere tested in ELISA with directly coated antigen or indirectly capturedbiotinylated antigen via immobilised biotinylated BSA-streptavidin.Tetanus toxoid and phOx-BSA were coated at 10 μg/ml in 0.1 M NaHCO₃ pH9.6 for 16 h at 4° C. For coating of hCG and hFSH-CTP a concentration of4 μg/ml in 50 mM NaHCO₃ pH 9.6 was used. For capture of biotinylatedantigens, biotinylated BSA was coated at 2 μg/ml in PBS during 1 h at37° C. After 3 washes with PBS-0.1% (v/v) Tween 20 (PBST), plates wereincubated during 1 h with streptavidin (10 μg/ml in PBS/0.5% gelatin)(Henderikx, et al., (1998) Cancer Res. 58, 4324-4332). Following washingas above, biotinylated antigen was added for an overnight incubation at4° C. at a concentration of 0.5 μg/ml for MUC-1 peptide, 3 μg/ml forhLH, and 0.6 μg/ml for hFSH (binding to hCG was tested with directlycoated antigen). The plates were blocked during 30 min at roomtemperature with 2% (w/v) semi-skimmed milk powder (Marvel) in PBS. Theculture supernatant was diluted 1 or 5-fold in 2% (w/v) Marvel/PBS andincubated 2 h; bound Fab was detected with anti-myc antibody 9E10 (5μg/ml) recognising the myc-peptide tag at the carboxyterminus of theheavy Fd chain, and rabbit anti-mouse-HRP conjugate (DAKO) (Marks, etal., (1991) J. Mol. Biol. 222, 581-597). Following the last incubation,staining was performed with tetramethylbenzidine (TMB) and H₂O₂ assubstrate and stopped by adding half a volume of 2 N H₂SO₄; the opticaldensity was measured at 450 nm. Clones giving a positive signal in ELISA(over 2× the background), were analysed by BstNI-fingerprinting of thePCR-products obtained by amplification with the oligonucleotide primersM13-reverse and geneIII-forward (Marks, et al., (1991) J. Mol. Biol.222, 581-597).

Large-scale induction of soluble Fab fragments from individual cloneswas performed on 50 ml scale in 2×TY containing 100 μg/ml ampicillin and2% glucose. After growth at 37° C. to an OD₆₀₀ of 0.9, the cells werepelleted (10 min at 2,934×g) and resuspended in 2×TY with ampicillin and1 mM IPTG. Bacteria were harvested after 3.5 h growing at 30° C. bycentrifugation (as before); periplasmic fractions were prepared byresuspending the cell pellet in 1 ml ice cold PBS. After 2 to 16 hrotating head-over-head at 4° C., the spheroplasts were removed by twocentrifugation steps: after spinning during 10 min at 3,400×g, thesupernatant was clarified by an additional centrifugation step during 10min at 13,000×g in an eppendorf centrifuge. The periplasmic fractionobtained was directly used for determination of fine specificities bysurface plasmon resonance or for western blot studies.

For sequencing, plasmid DNA was prepared from 50 ml cultures grown at30° C. in LB-medium, containing 100 μg/ml ampicillin and 2% glucose,using the QIAGEN midi-kit (Qiagen). Sequencing was performed with thethermocycling kit (Amersham) with CY5-labeled primers CH1FOR (5′-GTC CTTGAC CAG GCA GCC CAG GGC-3′-SEQ ID NO: 3) and M13REV (5′-CAG GAA ACA GCTATG AC-3′-SEQ ID NO: 4); samples were run on the ALF-Express(Pharmacia). V-gene sequences were aligned to V-base (Tomlinson et al.,V-BASE, MRC Centre for Protein Engineering, 1997,http://www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html) or the SangerCentre (Sanger Centre Germline Query, 1997, http//www.sanger.ac.uk/DataSearch/gq-search.html).

An hCG-preparation purified from urine and immuno-affinity purifiedrecombinant hLH, hFSH and hFSH-CTP produced in CHO-cells (Matzuk, etal., (1989) J. Cell. Biol. 109, 1429-1438; Muyan, et al., (1996) Mol.Endocrinol. 10, 1678-1687) were used for western blot studies as wasdescribed (Moyle, et al., (1990) J. Biol. Chem. 265, 8511-8518). Between0.5 and 1 μg of each hormone was loaded per lane; proteins were dilutedin non-reducing sample buffer and boiled during 5 min or directlyapplied on gel without heat-treatment; proteins were transferred toblotting membrane by electrotransfer. Blots were subsequently incubatedfor 16 h at room temperature with a 10-fold diluted periplasmic fractionin PBS/4% Marvel. Bound Fab was detected with anti-myc antibody 9E10 (5μg/ml) and 4,000-fold diluted anti-mouse alkaline phosphatase-conjugate(Promega), using the substrates 5-bromo-1-chloro-3-indolyl phosphate(BCIP) and nitro blue tetrazolium (NBT) (Boehringer Mannheim) forvisualisation.

The specificity of the Fab's was further characterised by surfaceplasmon resonance (BIAcore 2000, Biacore). Recombinant hLH, hFSH and theurinary hCG were immobilised on the flow-cells of a CM-chip using theNHS/EDC-kit (Pharmacia), yielding a surface of 1906 RU for hLH, 1529 RUfor hFSH and 1375 RU for hCG. Periplasmic fractions were dilutedthree-fold in Hepes Buffered Saline (HBS; 10 mM Hepes, 3.4 mM EDTA, 150mM NaCl, 0.05% (v/v) surfactant P20, pH 7.4) and analysed using a flowrate of 10 μl/min.

Fab's were obtained by refolding of the total bacterial proteins from a50 ml culture (de Haard, et al., (1998) Protein Eng., 11:1267-1276).Briefly, the pelleted cells from a 50 ml induced bacterial culture wereresuspensed in 8 ml 8 M urea (in PBS). After sonication, the mixture wasrotated head over head for 30 min and insoluble material was removed bycentrifugation for 30 min at 13,000×g. The supernatant was dialysedagainst PBS with four buffer changes. Insoluble proteins were removed bycentrifugation and the flow through fraction, obtained by filtrationthrough a 0.2 μm membrane, was immediately loaded on an hCG column (bedvolume 0.3 ml). The column material was prepared by coupling 8.4 mgprotein to one gram Tresyl sepharose according to the suppliersinstructions (Pierce). The column (1 ml column material) was washed with10 volumes 100 mM Tris, 500 mM NaCl pH 7.5, subsequently with 10 volumes100 mM Tris/500 mM NaCl pH 9.5 and with 2 volumes 0.9% NaCl, bound Fabwas eluted with two volumes 0.1 M TEA and immediately neutralised with0.5 volume 1M Tris pH 7.5. The Fab fraction was dialysed against PBSusing a Microcon 30 spin dialysis filter (Amicon). Finally, agel-filtration analysis was carried out on a Superdex 75HR column(Pharmacia). The yield was determined by measuring the optical densityat 280 nm (using a molar extinction coefficient of 13 for Fab's).

The kinetics of binding were analysed by surface plasmon resonance onthree different hCG surfaces (303 RU, 615 RU and 767 RU immobilised,with 4955 RU BSA on a separate flow cell as a negative control). Fabpresent in crude periplasmic extracts was quantified on a high densitysurface of purified anti-human-Fab polyclonal antibody (Pierce) asdescribed (Kazemier, et al., (1996) J. Immunol. Methods 194, 201-209).Anti-hCG Fab's controls were purified by affinity chromatography on hCGcolumns as described above and used to calibrate the system.

The Fab library was constructed in two-steps. In the first step,variable region gene pools were amplified from approx. 4×10⁸ B-cellsfrom the PBLs of four healthy donors, and, as a source of possibly moreheavily mutated IgM antibodies, from a segment of a (tumor-free) spleenremoved from a patient with gastric carcinoma, containing approximately1.5×10⁸ B-cells (Roit, et al., (1985) Immunology, Gower MedicalPublishing, Ltd., London). Only IgM-derived V_(H) segments wereamplified by using an amplification with an oligonucleotide primerlocated in the first constant domain of this isotype. These productswere cloned into phagemid vector pCES1 for V_(L), and in pUC119-CES1 forV_(H) (cloning was more efficiently in the smaller sized vector, inwhich gene III was deleted). The PBL and spleen derived V_(H), V_(K) andV_(λ)-libraries were cloned separately to maintain diversity, to yieldone-chain libraries in size typical for libraries made by cloning ofPCR-fragments (Marks, et al., (1991) J. Mol. Biol. 222, 581-597):1.75×10⁸ individual clones for the heavy chain, 9.4×10⁷ clones forV_(K), and 5.2×10⁷ clones for V_(λ). In the second step, the heavy chainfragments were digested from plasmid DNA isolated from the primary V_(H)repertoire, and cloned into the vector containing the light chainrepertoires (again separately for PBL and spleen derived repertoire).The libraries were combined using this efficient cloning procedure, tocreate a naVve Fab repertoire with 3.7×10¹⁰ individual clones (4.3×10¹⁰recombinant clones, 86% of which have a full-length Fab insert), with70% of clones harbouring a kappa light chain, 30% a lambda chain. All of20 clones with full length Fab insert tested scored positive in dot-blotanalysis with the 9E10 antibody to indicating an expression level ofsoluble Fab of at least 0.2 mg/L.

We evaluated the library by selection with different antigens. First,the results from three model antigens, the protein tetanus toxoid, thehapten 2-phenyloxazol-5-one (phOx) (Griffiths, et al., (1984) Nature312, 271-275, and the peptide MUC1, are discussed. Three rounds ofbiopanning on tetanus toxoid yielded a diverse set of ELISA positiveFab's, in a series of 47 tetanus toxoid binding Fab's, at least 21 weredifferent with regard to BstNI-fingerprint. Similarly, an extensivepanel of phOx-specific Fab's was retrieved after three rounds ofpanning: at least 24 different clones were identified in a series of 50ELISA positive clones. Solution capture with biotinylated MUC1 peptideresulted in the selection of 14 different antibody fragments out of 37ELISA-positive clones selected after 3 rounds.

As a more stringent test panel of antigens to assay the performance ofthe library, we chose to derive antibodies to three structurally relatedglycoproteins: human Chorionic Gonadotropin (hCG), human LuteinizingHormone (hLH) and human Follicle Stimulating Hormone (hFSH) (reviewed in(Cole, (1997) Clin. Chem. 43, 2233-2243)). These hormones areheterodimers sharing an identical α-chain with 92 amino acid residues,but have β-subunits of different composition and length. The β-chain ofhCG contains 145 amino acid residues, and the one from hLH only 121residues, the latter showing 85% homology to β-hCG. The β-chain of hFSHis only 111 amino acids and shares 36% of the residues with hCG.Antibodies that specifically detect hCG have been used extensively inpregnancy tests (Cole, (1997) Clin. Chem. 43, 2233-2243) and for cancerdiagnosis (Masure, et al., (1981) J. Clin. Endocrinol. Metab. 53,1014-1020; Papapetrou, et al., (1980) Cancer 45, 2583-2592). A large setof antibodies to these targets would extend the limited number ofhormone specific antibodies (especially against hLH), obtained using thehybridoma technology (Cole, (1997) Clin. Chem. 43, 2233-2243). The humanorigin of the antibodies might be beneficial when using these forimaging or therapy of testicular and bladder cancer (Masure, et al.,(1981) J. Clin. Endocrinol. Metab. 53, 1014-1020; Papapetrou, et al.,(1980) Cancer 45, 2583-2592).

Selections were thus performed on biotinylated urinary hCG, recombinanthLH, hFSH and hFSH-CTP (the latter is a chimeric molecule containing thecarboxy terminal peptide of β-hCG fused to the β-chain of FSH (Fares, etal., (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4304-4308)). The highestdegree of enrichment in respect to the increase in the number of elutedphage particles in round 3 versus round 1 was found for hCG(10,000-fold), followed by hFSH-CTP (1,000-fold), hFSH (300-fold) andhLH (150-fold). Polyclonal phage of selected populations were tested forbinding using sensorchips containing immobilised hormones (Schier, etal., (1996) Hum. Antibodies Hybridomas 7, 97-105). Polyclonal phageselected with hCG showed binding after rounds two and three of selectionto all three proteins, i.e., hCG, hLH, and hFSH, with the strongestsignal visible for hCG. Similar analysis of the polyclonal phagepopulations selected for three rounds on hFSH showed a dominance ofhFSH-specific binding, while selections on hFSH-CTP yielded binders toboth hFSH and hCG. Selections on hLH yielded antibodies reactive withhFSH and hCG. Thus, this polyclonal phage screening provides a rapidtest to check the overall quality of the clones in the selectedrepertoire, and may also be used to guide the choice of the conditionsfor the next selection round (Schier, et al., (1996) Hum. AntibodiesHybridomas 7, 97-105).

ELISA of monoclonal phage antibodies revealed that three rounds ofselection with hCG indeed resulted in the isolation of a high percentage(74%) of clones positive for the gonadotropin. 27% of these clones werehLH cross-reactive; none were reactive against streptavidin.BstNI-fingerprint analysis of the ELISA-positive clones revealed a highdegree of diversity (8 different patterns). From a representativehCG-specific (coded CG#4F) and hLH cross-reactive (CG#5C) clone, thespecificity was tested in BIAcore using unpurified soluble Fabfragments. Clone CG#4F gave a high response on hCG, with no visiblebinding to either hLH or hFSH-CTP. In contrast, clone CG#5C bound to hCGand hLH, but not to hFSH-CTP. Western blots, with the different hormonesin non-reduced form, showed the specific recognition of the β-subunit ofhCG by clone CG#4F, while the cross-reactive clone CG#5C reacted withthe β-subunit of both hCG and hLH.

Selection with the hormone hLH resulted in the isolation of hLH-specificand hCG cross-reactive clones. Examination of individual clones fromselection round three in ELISA revealed a large fraction of hLH specificclones (69%), and a minor group of cross-reactive clones (16%); nostreptavidin reactive clones were selected. Within the group of specificclones, a large array of different species (>21) could be discriminatedwith fingerprint analysis; however, all cross-reactive species had asingle pattern. The unique hLH specificity was confirmed forrepresentative clones LH#2H and LH#3G, shown in surface plasmonresonance; and on western blot. LH#3G only recognises the intactα/β-heterodimer of hLH. Two representative clones of a pan-reactiveantibody in ELISA, coded LH#1C and LH#3F, reacted in BIAcore withhFSH-CTP, hCG and hLH, and in western blot analysis with the α-chainsfrom all three hormones.

When hFSH was used as antigen during selection, 6 different antibodieswere isolated from the library, with one type, represented by cloneFS#8B, dominating the selected population. This Fab only recognised hFSHin BIAcore, and, as western blot analysis demonstrated, in particularits β-unit. Further, the specificity of an α-chain binding clone, SC#2B,was confirmed in BIAcore and western blot.

Upon selection with FSH-CTP 7 different α-chain specific Fab's wereidentified by fingerprint analysis, from which the clones coded SC#2B,SC#2F, SC#2G and SC#4G were examined in more detail. Immunoblot analysiswith the recombinant Fab as detecting antibody confirmed the α-chainspecificity.

The affinities and off-rates of affinity purified hCG reactive Fab'sLH#1C, SC#2B, LH#3F and CG#5C were determined. The off-rates for mostFab's were in the order of 10⁻² and 10⁻³ s⁻¹. The off-rate valuesobtained using crude periplasmic fractions were in good agreement withthe values found for the purified Fab's, validating the utility of theoff-rate screen with unpurified Fab fragments. The affinities, 23 nM and38 nM for the α-subunit specific antibody LH#1C and the β-subunithCG/hLH-cross reactive antibody CG#5C respectively, are comparable tothe affinity of antibodies selected from a murine immune phage antibodylibrary (H.d.H., B. Kazemier, et al., unpublished); the top affinity,2.7 nM for the α-chain specific Fab SC#2B, approaches the values of thebest anti-hCG monoclonal antibodies (H.d.H., B. Kazemier, et al.,unpublished).

The aim of this procedure is to select and enrich for phage-antibodiesto an antigen coated on the surface of immunotubes. The antigen iscoated to the immunotube (e.g., a Nunc-immunotube) and incubated withthe phage library. Non-bound phage are washed away and the binding phageare eluted, therefore the phage library becomes enriched for phageantibodies that specifically bind the antigen.

The aim of this procedure is to biotinylate proteins or peptides. Atneutral pH or above, primary amine-groups react with NHS-SS-Biotin, andN-hydroxysulfosuccimide is released. The N-terminal free NH₂-groups aswell as lysines (K) of the protein react with NHS-S-S-Biotin, in this pHrange.

NHS-SS-Biotin is a unique biotin analog with an extended spacer arm ofapproximately 24.3 Å in length, the spacer arm of NHS-LC-Biotin is 22.4Å. These long chain analogs reduce steric hindrances associated withbinding of biotinylated molecules to avidin or streptavidin.

The presence of the S-S linker in NHS-S-S-Biotin enables disruption ofbinding using reducing agents (DTT, DTE, B-mercaptoethanol).NHS-LC-Biotin is used when biotinylated protein/peptide is needed thatis not sensitive to reducing agents.

The aim of this procedure is to select phage antibodies against abiotinylated antigen. The selection is done in solution, and can be usedto select phage antibodies against antigens that are prone todenaturation when coated onto solid surfaces.

First the biotinylated antigen is incubated with the phage antibodylibrary. After addition of the Dynabeads (Dynal) coated withstreptavidin, the biotin of the antigen-antibody-complex will bind tothe streptavidin. This Dynabead-antigen-antibody-complex is pulled outwith a magnet (e.g., a Dynal magnet) and therefore should contain thespecific antibodies.

The aim of this procedure is to select for those antibodies out of alibrary that bind to antigens present in the cell membrane, usingadherent growing cells or cells in suspension. The method can be usedfor selection of antibodies against targets expressed on (tumor) celllines.

By incubating whole cells, organelles, or membrane fractions with a highvariety phage antibody repertoire, such as the Fab Libraries of theinvention (concentrated by PEG precipitation), only (or preferentially)relevant antibodies, to one of the molecules exposed on the surface ofthe cellular membrane(s), will be retained while not binding phageantibodies are separated from the antibodies bound to the cells,organelles or membrane fractions (by methods well known in the art forseparating cells, organelles or membrane fractions from molecules insolution). The retained phage population is enriched for those cloneswhich are specific for cell related molecules. In principle thefollowing factors will positively influence the enrichment of individualclones: Affinity, antigen abundance, and low toxicity of the antibodyconstruct to TG1 host.

The aim of this procedure is to prepare soluble antibody fragments fromthe periplasm of E. coli. In the periplasm there is: less proteaseactivity, less contaminating proteins than in the cytoplasm orsupernatants, and the antibody is more concentrated. Therefore,periplasmic preparations are more stable and more pure than culturesupernatants.

As a consequence of induction of phagemid containing bacterial culturesin low glucose medium with IPTG, soluble antibody fragments are producedand directed to the periplasm where they are concentrated within 4hours. Overnight culturing in these circumstances will make thebacterial membrane leaky and antibodies will be found in thesupernatant. For preparation of periplasmic fractions, the bacterialcell wall is first lysed by cold osmotic shock (icecold TES) and thenrapidly diluted in a chilled solution of low osmotic strength (TES/H₂O).The EDTA makes the outer membrane more permeable, and the cold inhibitsprotease activity. Subsequently, the bacterial cells are spun down andthe supernatant then contains the periplasmic proteins.

The antibodies in the periplasmic fraction can be used as a ‘crudeextract’ or the antibodies can be purified by conventional means wellknown in the art, for e.g., those recited in Section 5.6 and 5.7.

The aim of this procedure is to purify antibodies labeled with a His6tag from periplasmic fractions of Fabs made as described in Example6.18.

Immobilized metal affinity chromatography (IMAC) for the purification ofrecombinant 6×His-tagged proteins under native conditions: Recombinanthistidine tagged proteins are captured on a chelated metal containingresin through coordination of free N-atoms of the histidines to themetal (mostly Ni²⁺ or Co²⁺). After washing away contaminating proteinsand other cell constituents, the his-tagged protein is specificallyeluted from the resin with imidazol which competes for the binding ofhistidine-residues to the metal ion.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention, and compositions and methods which are functionallyequivalent are within the scope of the invention. Indeed, numerousmodifications and variations in the practice of the invention areexpected to occur to those skilled in the art upon consideration of thepresent preferred embodiments. Consequently, the only limitations whichshould be placed upon the scope of the invention are those which appearin the appended claims.

All references cited within the body of the instant specification arehereby incorporated by reference in their entirety.

1-7. (canceled)
 8. A method for making a Fab library, comprising:providing a plurality of vectors, each comprising (a) a first cloningregion, (b) a second cloning region, wherein each cloning region of (a)and (b) comprises at least one, for the vector unique, restrictionenzyme cleavage site, and each cloning region is 5′ flanked by aribosome binding site and a signal sequence (c) a polynucleotideencoding an anchor region located 3′ of the second cloning region, and(d) a polynucleotide encoding a heavy chain constant region or a portionthereof located between the vector unique restriction enzyme cleavagesite of the second cloning region and the anchor region; introducinginto the first coding region a member of a first plurality of variablepolynucleotides encoding a first plurality of variable polypeptides,each of which comprises an antibody light chain variable region, andseparately introducing into the second cloning region of each vector amember of a second plurality of variable polynucleotides encoding asecond plurality of variable polypeptides, each of which comprises anantibody heavy chain variable region, thereby producing a plurality ofvectors encoding a plurality of Fab molecules.
 9. The method of claim 8,wherein each of the vectors further comprises a polynucleotide encodinga light chain constant region located 3′ of the first cloning region.10. The method of claim 8, wherein each of the first plurality ofvariable polypeptides further comprises a light chain constant regionfused to the C-terminus of the light chain variable region.
 11. Themethod of claim 1, further comprising introducing the plurality ofvectors into host cells to provide a plurality of capsid particles, eachcomprising a member of the first plurality of variable polynucleotidesand a member of the second plurality of variable polynucleotides,wherein the polypeptides encoded by the members of the first and secondplurality of variable polynucleotides form a Fab molecule.
 12. Themethod of claim 1, wherein the plurality of vectors in the libraryencode at least 10⁹ different Fab molecules.
 13. The method of claim 12,wherein the plurality of vectors in the library encode at least 10¹⁰different Fab molecules.
 14. The method of claim 13, wherein theplurality of vectors in the library encode at least 3.7×10⁹ differentFab molecules.
 15. The method of claim 11, wherein the plurality ofcapsid particles comprise at least 10⁹ different Fab molecules.
 16. Themethod of claim 15, wherein the plurality of capsid particles compriseat least 10¹⁰ different Fab molecules.
 17. The method of claim 16,wherein the plurality of capsid particles comprise at least 3.7×10¹⁰different Fab molecules.
 18. The method of claim 1, further comprising:amplifying the first plurality of variable polynucleotides with a firstset of primers, and amplifying the second plurality of variablepolynucleotides with a second set of primers, wherein each set of theprimers comprises oligonucletodies are homologous to the 5′ and 3′ endof polynucleotides encoding antibody variable regions or parts thereoffor amplifying variable polynucleotide pools from natural or syntheticsources of antibody genes.
 19. The method of claim 1, wherein the anchorregion comprises a phage coat protein.
 20. The method of claim 19,wherein the coat protein is a gene III product.
 21. The method of claim20, wherein the coat protein comprises minor coat protein III offilamentous phage f_(d).